Rationally designed media for cell culture

ABSTRACT

This invention relates to methods for rationally designing cell culture media for use in cell cultures, e.g., cell cultures employed in polypeptide production; cell culture media designed with the disclosed methods; methods of producing a polypeptide of interest, e.g., an antibody, using such media; polypeptides produced using the methods and media disclosed herein; and pharmaceuticals compositions containing such polypeptides. The rationally designed media contain a concentration of an amino acid that is calculated for use in cell mass, a concentration of the amino acid that is calculated for use in cell maintenance, and a concentration of the amino acid that is calculated for incorporation into the polypeptide of interest.

RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalPatent Application No. 60/858,289, filed Nov. 8, 2006, the content ofwhich is hereby incorporated by reference herein in its entirety

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for rationally designing cell culturemedia for use in cell cultures employed in, e.g., polypeptideproduction; cell culture media designed with the disclosed methods;methods of producing large quantities of a polypeptide of interest,e.g., an antibody, using such media; polypeptides produced using themethods and media disclosed herein; and pharmaceuticals compositionscontaining such polypeptides. The invention is particularly useful inlarge-scale cell cultures. The methods and compositions disclosed hereinare particularly useful to produce significant quantities ofpolypeptides in batch, fed-batch and perfusion animal cell cultures.

2. Related Background Art

A large proportion of biotechnology products, whether commerciallyavailable or only in development, are protein therapeutics; thus, thereis a demand for production of these polypeptides in cell cultures.Furthermore, the cellular machinery of an animal cell (as opposed to,e.g., a bacterial cell) is often required to produce many forms ofpolypeptide therapeutics (such as glycosylated proteins orhybridoma-produced monoclonal antibodies (MAbs)). Consequently, there isan increasing demand for optimizing production of these polypeptides incell cultures, and particularly in animal cell cultures.

As compared to bacterial cell cultures, animal cell cultures have lowerproduction rates and typically generate lower production yields. Thus, asignificant quantity of research focuses on animal cell cultureconditions that optimize the polypeptide output, i.e., conditions thatsupport high cell density and high titer. For example, it has beendetermined that maintaining glucose concentrations in cell culture mediaat low concentrations and culturing cells in a production phase at anosmolality of about 400 to 600 mOsm increases production of recombinantproteins by animal cell cultures, wherein culturing in all phases isalso at a selected glutamine concentration (preferably between about 0.2to about 2 mM). It has also been determined that restricted feeding ofglucose to animal cell cultures in fed-batch processes controls lactateproduction without requiring the constant-rate feeding of glucose.Further, it is known that modification of the total cumulativeconcentration of amino acids, the concentration of individual aminoacids, and the ratios of individual amino acids to each other (e.g.,glutamine to asparagine) and to total amino acids (e.g., glutamine tototal amino acids) in the media of a large-scale cell culture can resultin substantially improved large-scale polypeptide production.

Traditionally, medium studies for animal cell cultures focus on threetechniques: 1) enriching the medium components of the starting mediumand increasing the frequency of culture feeding; 2) applyingmulti-factorial design to different medium strengths and differentcomponent concentrations; and 3) analyzing conditioned (spent) mediumfor amino acids, vitamins, and other components, and adding thosecomponents that are at low levels or are depleted. These methodsgenerally use cell density, viability and titer responses as indicatorsof optimization.

However, the above methods only indirectly detect the nutrientrequirement for cells based on the end result, i.e., cell density,viability, and titer, rather than detecting and providing the cell withthe actual nutrient requirement for optimized protein production.

SUMMARY OF THE INVENTION

The present invention provides methods for rationally designing cellculture media, e.g., large-scale cell culture media, for use in, e.g.large-scale cell cultures employed in polypeptide production; cellculture media, e.g., large-scale cell culture media, designed with thedisclosed methods; methods of producing large quantities of apolypeptide of interest, e.g., an antibody, using such media;polypeptides produced using the methods and media disclosed herein; andpharmaceuticals compositions containing such polypeptides. These methodsand compositions are useful for culturing, e.g., batch, fed-batch, andperfusion culturing, of cells. These methods and compositions areparticularly useful for large-scale culturing, e.g., batch, fed-batch,and perfusion culturing, of animal cells, e.g., mammalian cells.

A rationally designed medium of the present invention contains aconcentration of an amino acid that is calculated for use in cell mass,a concentration of the amino acid that is calculated for use in cellmaintenance, and a concentration of the amino acid that is calculatedfor incorporation into the polypeptide of interest.

In one embodiment, the invention provides a method of producing apolypeptide in a cell culture comprising providing a cell culture,comprising cells, comprising a nucleic acid encoding a polypeptide ofinterest, and a desired cell culture medium, comprising a concentrationof an amino acid that is calculated for use in cell mass, aconcentration of the amino acid that is calculated for use in cellmaintenance, and a concentration of the amino acid that is calculatedfor incorporation into the polypeptide of interest; and, maintaining thecell culture under conditions that allow expression of the polypeptideof interest. In one embodiment of the invention, the desired cellculture medium comprises a baseline-adjusted amino acid concentration,A, according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is aconcentration of the amino acid that is used per unit of cell mass, P isa concentration of the amino acid that is used for incorporation intothe polypeptide of interest per unit of polypeptide titer, M is amultiplier for a desired peak cell density of the cell culture, N is amultiplier for a desired concentration of the polypeptide of interest, Yis a cell maintenance factor, and F is a baseline factor.

In another embodiment, the invention provides a method of producing apolypeptide in a cell culture, comprising providing a cell culture,comprising cells, comprising a nucleic acid encoding a polypeptide ofinterest; and a starting cell culture medium, wherein the volume of thestarting cell culture medium is about 60-99% of the volume of a desiredcell culture medium volume; providing a feeding cell culture medium tothe cell culture, wherein the volume of the feeding cell culture mediumis about 1-40% of the desired cell culture medium volume, and whereinthe resulting desired cell culture medium comprises a concentration ofan amino acid that is calculated for use in cell mass, a concentrationof the amino acid that is calculated for use in cell maintenance, and aconcentration of the amino acid that is calculated for incorporationinto the polypeptide of interest; and, maintaining the cell cultureunder conditions that allow expression of the polypeptide of interest.In one embodiment of the invention, the resulting desired cell culturemedium comprises a baseline-adjusted amino acid concentration, A,according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is aconcentration of the amino acid that is used per unit of cell mass, P isa concentration of the amino acid that is used for incorporation intothe polypeptide of interest per unit of polypeptide titer, M is amultiplier for a desired peak cell density of the cell culture, N is amultiplier for a desired concentration of the polypeptide of interest, Yis a cell maintenance factor, and F is a baseline factor; andmaintaining the cell culture under conditions that allow expression ofthe polypeptide of interest. In another embodiment of the invention, thestarting cell culture medium comprises a concentration, B, of the aminoacid according to the formula B=[A−(Z*V)]/(1 V), wherein Z is aconcentration of the amino acid in the feeding cell culture medium, andV is a volume of the feeding culture medium as a proportion of thedesired cell culture medium volume. In another embodiment of the methodsdisclosed herein, Y is 0 to about 1.5. In yet another embodiment of themethods disclosed herein, F is about 1 to about 1.5. In a furtherembodiment of the methods disclosed herein, Y is 0 to about 1.5 and F isabout 1 to about 1.5.

In one embodiment of the methods disclosed herein, the desired cellculture medium comprises greater than or equal to about 3 mM tyrosine.In another embodiments of the methods disclosed herein, the desired cellculture medium comprises: between about 7 mM and about 30 mM leucine;between about 7 mM and about 30 mM lysine; between about 7 mM and about30 mM threonine; between about 7 mM and about 30 mM proline; and/orbetween about 7 mM and about 30 mM valine. In a further embodiment ofthe methods disclosed herein, the combined concentration of leucine,lysine, threonine, proline, and valine in the desired cell culturemedium is between about 35 mM and about 150 mM. In yet anotherembodiment, the combined concentration of leucine, lysine, threonine,and valine in the desired cell culture medium is between about 60% andabout 80% of the concentration of the total essential amino acids in thedesired cell culture medium.

In one embodiment of the methods disclosed herein, the combinedconcentration of the essential amino acids in the desired cell culturemedium is between about 30% and about 50% of the concentration of thetotal amino acids in the desired cell culture medium. In anotherembodiment of the methods disclosed herein, the concentration of aminoacids in the desired cell culture medium is between about 120 mM andabout 350 mM. In a further embodiment of the methods disclosed herein,the concentration of praline in the cell culture is maintained atgreater than about 1 mM. In yet another embodiment of the methodsdisclosed herein, the concentration of proline in the cell culture ismaintained at greater than about 2 mM. In some embodiments of themethods of producing a polypeptide, the cell culture is a large-scalecell culture. In other embodiments, the cells are animal cells.

A further aspect of the invention provides polypeptides producedaccording to the methods disclosed herein. Another aspect of theinvention provides a pharmaceutical composition comprising a polypeptideproduced according to the methods disclosed herein and apharmaceutically acceptable carrier.

A further aspect of the invention provides a method of cell culturecomprising: providing a cell culture, comprising: cells; and a desiredcell culture medium, comprising a concentration of an amino acid that iscalculated for use in cell mass and a concentration of the amino acidthat is calculated for use in cell maintenance; and maintaining the cellculture under conditions that allow growth and replication of the cellsin the cell culture. In one embodiment of the invention, the desiredcell culture medium comprises a baseline-adjusted amino acidconcentration, A′, according to the formula A′=[(M*X)+(Y*M*X)]*F,wherein X is a concentration of the amino acid that is used per unit ofcell mass, M is a multiplier for a desired peak cell density of the cellculture, Y is a cell maintenance factor, and F is a baseline factor. Insome embodiments of the methods of cell culture, the cell culture is alarge-scale cell culture. In other embodiments, the cells are animalcells.

A further aspect of the invention provides a cell culture medium,comprising a total concentration of amino acids from between about 120mM and about 350 mM. Another aspect of the invention provides a cellculture medium for use in the production of a polypeptide of interest,comprising a total concentration of amino acids from between about 120mM and about 350 mM.

Another aspect of the invention provides a cell culture medium for usein the production of a polypeptide of interest, comprising aconcentration of an amino acid that is calculated for use in cell mass,a concentration of the amino acid that is calculated for use in cellmaintenance, and a concentration of the amino acid that is calculatedfor incorporation into the polypeptide of interest. In one embodiment ofthe invention, the cell culture medium for use in the production of apolypeptide of interest comprises a baseline-adjusted amino acidconcentration, A, according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F,wherein X is a concentration of the amino acid that is used per unit ofcell mass, P is a concentration of the amino acid that is used forincorporation into the polypeptide of interest per unit of polypeptidetiter, M is a multiplier for desired peak cell density of the cellculture, N is a multiplier for desired concentration of the polypeptideof interest, Y is a cell maintenance factor, and F is a baseline factor.

Yet another aspect of the invention provides a cell culture medium,comprising a baseline-adjusted amino acid concentration, A′, accordingto the formula A′=[(M*X)+(Y*M*X)]*F, wherein X is a concentration of theamino acid that is used per unit of cell mass, M is a multiplier fordesired peak cell density of the cell culture, Y is a cell maintenancefactor, and F is a baseline factor. In some embodiments, the cellculture medium is a large-scale cell culture medium. In otherembodiments, the cell culture medium is an animal cell culture medium.

Yet another aspect of the invention provides a method for determining anoptimized concentration of an amino acid used in a cell culture mediumfor the production of a polypeptide of interest in a cell culture,comprising: determining the amino acid concentration required for thecell mass of the cells in the cell culture at a target cell density;determining the amino acid concentration required to produce thepolypeptide of interest in the cell culture at a target polypeptidetiter; determining the amino acid concentration required for cellmaintenance of the cells in the cell culture; and adding theconcentrations to provide an optimized concentration of the amino acidused in the cell culture medium for the production of the polypeptide ofinterest in the cell culture.

A further aspect of the invention provides a method for determining anoptimized amino acid concentration, A, of an amino acid used in a cellculture medium for the production of a polypeptide of interest in a cellculture, comprising: determining the amino acid concentration, X,required for cell mass of the cells at a set cell density; determiningthe amino acid concentration, P, required to produce the polypeptide ofinterest at a set polypeptide titer; and determining the optimized aminoacid concentration, A, according to the formulaA=[(M*X)+(N*P)+(M*Y*X)]*F, wherein M is a multiplier for a desiredtarget cell density of the cell culture, N is a multiplier for a desiredtarget concentration of the polypeptide of interest, Y is a cellmaintenance factor; and F is a baseline factor. In some embodiments ofthe methods for determining an optimized amino acid concentration, thecell culture is a large-scale cell culture. In other embodiments, thecells are animal cells.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cell density (Y-axis; “Cell Density (10⁶ cells/mL)”)over time (X-axis; “Days”) for CHO cells engineered to expressanti-IL-22. Cells were cultured in the rationally designed medium ofExample 2.

FIG. 2 depicts the titer (Y-axis; “Titer (g/L)”) of anti-IL-22 antibodyover time (X-axis; “Days”) for CHO cells engineered to expressanti-IL-22. Cells were cultured in the rationally designed medium ofExample 2.

FIG. 3 depicts the cell density (Y-axis; “Cell Density (10⁶ cells/mL)”)over time (X-axis; “Days”) for CHO cells engineered to expressanti-IL-22. Cells were cultured in the rationally designed medium ofExample 3.

FIG. 4 depicts the titer (Y-axis; “Titer (g/L)”) of anti-IL-22 antibodyover time (X-axis; “Days”) for CHO cells engineered to expressanti-IL-22. Cells were cultured in the rationally designed medium ofExample 3.

FIG. 5 depicts the titer (Y-axis; “Titer (g/L)”) of anti-IL-22 antibodyover time (X-axis; “Days”) for CHO cells engineered to expressanti-IL-22. Cells were cultured in “Traditional Medium” (a medium basedon traditional cell culture requirements, see, e.g., U.S. PublishedPatent Application No. 2006/0121568), “Rational Design Medium” preparedusing the methods herein, or “Traditional Medium+Proline,” whichcontains an additional 3.7 mM proline added to the “Traditional Medium”(see Example 4).

FIG. 6 depicts the cell density (Y-axis; “Cell Density (10⁶ cells/mL)”over time (X-axis; “Days”) for CHO cells engineered to expressanti-IL-22. Cells were cultured in “Traditional Medium,” “RationalDesign Medium,” or “Traditional Medium+Proline” (see Example 4).

FIG. 7 depicts the cell viability (Y-axis; “Viability [%]”) over time(X-axis; “Days”) for CHO cells engineered to express anti-IL-22. Cellswere cultured in “Traditional Medium,” “Rational Design Medium,” or“Traditional Medium+Proline” (see Example 4).

FIG. 8 depicts the concentration of the designated amino acid (Y-axis;“[μM]”) ((FIG. 8A) proline; (FIG. 8B) threonine; (FIG. 8C) valine; (FIG.8D) tryptophan; or (FIG. 8E) tyrosine) over time (X-axis; “Days”) forCHO cells for cells engineered to express anti-IL-22. Cells werecultured in “Traditional Medium,” “Rational Design Medium,” or“Traditional Medium+Proline” (see Example 4).

FIG. 9 depicts the cell density (Y-axis; “Cell Density (10⁶ cells/mL)”)over time (X-axis; “Days”) for CHO cells engineered to expressanti-IL-22. Cells were cultured in “Rational Design Medium” or “RationalDesign Medium without maintenance and baseline factors.” The figure isrepresentative of 5 independent replicates (n=5) (see Example 6).

FIG. 10 depicts cell viability (Y-axis: “Viability (%)”) over time(X-axis; “Days”) for CHO cells engineered to express anti-IL-22. Cellswere cultured in “Rational Design Medium” or “Rational Design Mediumwithout maintenance and baseline factors.” The figure is representativeof 5 independent replicates (n=5) (see Example 6).

FIG. 11 depicts antibody titer (Y-axis; “Titer (g/L)”) over time(X-axis; “Days”) for CHO cells engineered to express anti-IL-22. Cellswere cultured in “Rational Design Medium” or “Rational Design Mediumwithout maintenance and baseline factors.” The figure is representativeof 5 independent replicates (n=5) (see Example 6).

DETAILED DESCRIPTION OF THE INVENTION

The term “batch culture” as used herein refers to a method of culturingcells in which all the components that will ultimately be used inculturing the cells, including the medium as well as the cellsthemselves, are provided at the beginning of the culturing process. Abatch culture is typically stopped at some point and the cells and/orcomponents in the medium are harvested and optionally purified.

The term “fed-batch culture” as used herein refers to a method ofculturing cells in which additional components are provided to theculture at some time subsequent to the beginning of the culture process.The provided components typically comprise nutritional supplements forthe cells that have been depleted during the culturing process. Afed-batch culture is typically stopped at some point and the cellsand/or components in the medium are harvested and optionally purified.In a preferred embodiment of the present invention, the cell culture isan animal cell culture, e.g., a mammalian cell culture, that is a batchor fed-batch culture.

The term “perfusion culture” as used herein refers to a method ofculturing cells in which additional components are provided continuouslyor semi-continuously to the culture subsequent to the beginning of theculture process. The provided components typically comprise nutritionalsupplements for the cells that have been depleted during the culturingprocess. Portions of the cells and/or components in the medium aretypically harvested on a continuous or semi-continuous basis and areoptionally purified.

The term “bioreactor” as used herein refers to any vessel used for thegrowth of a prokaryotic or eukaryotic cell culture, e.g., an animal cellculture (such as a mammalian cell culture). The bioreactor can be of anysize so long as it is useful for the culturing of cells, e.g., mammaliancells. Typically, the bioreactor will be at least 30 ml and may be 1,10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,0000 liters ormore, or any intermediate volume. The internal conditions of thebioreactor, including, but not limited to pH and temperature, aretypically controlled during the culturing period. The bioreactor can becomposed of any material that is suitable for holding mammalian cellcultures suspended in media under the culture conditions of the presentinvention, including glass, plastic or metal. The term “productionbioreactor” as used herein refers to the final bioreactor used in theproduction of the polypeptide or protein of interest. The volume of alarge-scale cell culture production bioreactor is generally greater thanabout 100 ml, typically at least about 10 liters, and may be 500, 1000,2500, 5000, 8000, 10,000, 12,0000 liters or more, or any intermediatevolume. One of ordinary skill in the art will be aware of, and will beable to choose, suitable bioreactors for use in practicing the presentinvention.

The terms “cell density,” “cell concentration,” or the like, as usedherein, refer to that number, weight, mass, etc. of cells present in agiven volume of medium. “Peak cell density” or the like refers to themaximum number of cells that can be reached in a given volume of medium,and “desired peak cell density” or the like refers to the maximum numberof cells that a practitioner desires to obtain (e.g., targets) in agiven cell volume. Variations of such target value(s) will be clear tothose of skill in the art, e.g., one of skill may express a targetvalue(s) in terms of desired cell mass, and such target value(s) may bein one or more appropriate units of measure (e.g., desired peak units ofcell mass).

The term “cell viability” as used herein refers to the ability of cellsin culture to survive under a given set of culture conditions orexperimental variations. The term as used herein also refers to thatportion of cells that are alive at a particular time in relation to thetotal number of cells, living and dead, in the culture at that time.

The terms “culture” and “cell culture” as used herein refer to a cellpopulation that is suspended in a cell culture medium under conditionssuitable to survival and/or growth of the cell population. As usedherein, these terms may refer to the combination comprising the cellpopulation (e.g., the animal cell culture) and the medium in which thepopulation is suspended.

The term “integrated viable cell density” or “IVC” as used herein refersto the average density of viable cells over the course of the culturemultiplied by the amount of time the culture has run. Assuming theamount of polypeptide and/or protein produced is proportional to thenumber of viable cells present over the course of the culture,integrated viable cell density is a useful tool for estimating theamount of polypeptide and/or protein produced over the course of theculture.

The terms “medium,” “cell culture medium,” and “culture medium” as usedherein refer to a solution containing nutrients that nourish growinganimal, e.g., mammalian, cells. Typically, these solutions provideessential and nonessential amino acids, vitamins, energy sources,lipids, and trace elements required by the cell for minimal growthand/or survival. The solution may also contain components that enhancegrowth and/or survival above the minimal rate, including hormones andgrowth factors. The solution is preferably formulated to a pH and saltconcentration optimal for cell survival and proliferation. In oneembodiment, the medium is a defined medium. Defined media are media inwhich all components have a known chemical structure. In anotherembodiment of the invention, the medium may contain an amino acid(s)derived from any source or method known in the art, including, but notlimited to, an amino acid(s) derived either from single amino acidaddition(s) or from peptone or protein hydrolysate (including animal orplant source(s)) addition(s).

The teen “seeding” as used herein refers to the process of providing acell culture to a bioreactor or another vessel. The cells may have beenpropagated previously in another bioreactor or vessel. Alternatively,the cells may have been frozen and thawed prior to, e.g., immediatelyprior to, providing them to the bioreactor or vessel. The term refers toany number of cells, including a single cell.

The term “titer” as used herein refers to the total amount ofpolypeptide of interest produced by an animal cell culture, divided by agiven amount of medium volume; thus “titer” refers to a concentration.Titer is typically expressed in units of milligrams of polypeptide permilliliter of medium.

As used herein, the term “antibody” includes a protein comprising atleast one, and typically two, VH domains or portions thereof, and/or atleast one, and typically two, VL domains or portions thereof. In certainembodiments, the antibody is a tetramer of two heavy immunoglobulinchains and two light immunoglobulin chains, wherein the heavy and lightimmunoglobulin chains are interconnected by, e.g., disulfide bonds. Theantibodies, or a portion thereof, can be obtained from any origin,including, but not limited to, rodent, primate (e.g., human and nonhumanprimate), camelid, etc., or they can be recombinantly produced, e.g.,chimeric, humanized, and/or in vitro-generated, e.g., by methods wellknown to those of skill in the art.

Examples of binding fragments encompassed within the term“antigen-binding fragment” of an antibody include, but are not limitedto, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH,CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment, which consists of a VH domain; (vi) acamelid or camelized heavy chain variable domain (VHH); (vii) a singlechain Fv (scFv; see below); (viii) a bispecific antibody; and (ix) oneor more fragments of an immunoglobulin molecule fused to an Fc region.Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv)); see, e.g., Bird et al.(1988) Science 242:423-26; Huston et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:5879-83). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding fragment” of an antibody.These fragments may be obtained using conventional techniques known tothose skilled in the art, and the fragments are evaluated for functionin the same manner as are intact antibodies.

The “antigen-binding fragment” can, optionally, further include a moietythat enhances one or more of, e.g., stability, effector cell function orcomplement fixation. For example, the antigen binding fragment canfurther include a pegylated moiety, albumin, or a heavy and/or a lightchain constant region.

Other than “bispecific” or “bifunctional” antibodies, an antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai and Lachmann (1990) Clin. Exp. Immunol. 79:315-21; Kostelnyet al. (1992) J. Immunol. 148:1547-53.

The phrase “protein” or “protein product” refers to one or more chainsof amino acids. As used herein, the term “protein” is synonymous with“polypeptide” and, as is generally understood in the art, refers to atleast one chain of amino acids liked via sequential peptide bonds. Incertain embodiments, a “protein of interest” or a “polypeptide ofinterest” is a protein encoded by an exogenous nucleic acid moleculethat has been transformed into a host cell. In certain embodiments,wherein the “protein of interest” is coded for by an exogenous DNA withwhich the host cell has been transformed, the nucleic acid sequence ofthe exogenous DNA determines the sequence of amino acids. In certainembodiments, a “protein of interest” is a protein encoded by a nucleicacid molecule that is endogenous to the host cell. In certainembodiments, expression of such an endogenous protein of interest isaltered by transfecting a host cell with an exogenous nucleic acidmolecule that may, for example, contain one or more regulatory sequencesand/or encode a protein that enhances expression of the protein ofinterest. Methods and compositions of the present invention may be usedto produce any protein of interest, including, but not limited toproteins having pharmaceutical, diagnostic, agricultural, and/or any ofa variety of other properties that are useful in commercial,experimental and/or other applications. In addition, a protein ofinterest can be a protein therapeutic. Namely, a protein therapeutic isa protein that has a biological effect on a region in the body on whichit acts or on a region of the body on which it remotely acts viaintermediates. Examples of protein therapeutics are discussed in moredetail below. In certain embodiments, proteins produced using methodsand/or compositions of the present invention may be processed and/ormodified. For example, a protein to be produced in accordance with thepresent invention may be glycosylated.

The present invention may be used to culture cells for the advantageousproduction of any therapeutic protein, such as pharmaceutically orcommercially relevant enzymes, receptors, antibodies (e.g., monoclonaland/or polyclonal antibodies), Fc fusion proteins, cytokines, hormones,regulatory factors, growth factors, coagulation/clotting factors,antigen binding agents, etc. One of ordinary skill in the art will beaware of other proteins that can be produced in accordance with thepresent invention, and will be able to use methods disclosed herein toproduce such proteins.

Expression Constructs and Generation of Recombinant Host Cells

The present invention uses recombinant host cells, e.g., prokaryotic oreukaryotic host cells, i.e., cells transfected with an expressionconstruct containing a nucleic acid that encodes a polypeptide ofinterest. The phrase “animal cells” encompasses invertebrate,nonmammalian vertebrate (e.g., avian, reptile and amphibian), andmammalian cells. Nonlimiting examples of invertebrate cells include thefollowing insect cells: Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori (silkworm/silk moth).

A number of mammalian cell lines are suitable host cells for recombinantexpression of polypeptides of interest. Mammalian host cell linesinclude, for example, COS, PER.C6, TM4, VERO076, MDCK, BRL-3A, W138, HepG2, MMT, MRC 5, FS4, CHO, 293T, A431, 3T3, CV-1, C3H10T1/2, Colo205,293, HeLa, L cells, BHK, HL-60, FRhL-2, U937, HaK, Jurkat cells, Rat2,BaF3, 32D, FDCP-1, PC12, M1x, murine myelomas (e.g., SP2/0 and NS0) andC2C12 cells, as well as transformed primate cell lines, hybridomas,normal diploid cells, and cell strains derived from in vitro culture ofprimary tissue and primary explants. Any eukaryotic cell that is capableof expressing the polypeptide of interest may be used in the disclosedmedia design methods. Numerous cell lines are available from commercialsources such as the American Type Culture Collection (ATCC). In oneembodiment of the invention, the cell culture, e.g., the large-scalecell culture, employs hybridoma cells. The construction ofantibody-producing hybridoma cells is well known in the art. In oneembodiment of the invention, the cell culture, e.g., the large-scalecell culture, employs CHO cells.

Alternatively, it may be possible to recombinantly produce polypeptidesof interest in lower eukaryotes such as yeast, or in prokaryotes such asbacteria. Suitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing polypeptide of interest. Suitable bacterialstrains include Escherichia coli, Bacillus subtilis, Salmonellatyphimurium, or any bacterial strain capable of expressing thepolypeptide of interest. Expression in bacteria may result in formationof inclusion bodies incorporating the recombinant protein. Thus,refolding of the recombinant protein may be required in order to produceactive or more active material. Several methods for obtaining correctlyfolded heterologous proteins from bacterial inclusion bodies are knownin the art. These methods generally involve solubilizing the proteinfrom the inclusion bodies, then denaturing the protein completely usinga chaotropic agent. When cysteine residues are present in the primaryamino acid sequence of the protein, it is often necessary to accomplishthe refolding in an environment that allows correct formation ofdisulfide bonds (a redox system). General methods of refolding aredisclosed in Kohno (1990) Meth. Enzymol. 185:187-95, EP 0433225, andU.S. Pat. No. 5,399,677.

The present invention uses constructs, in the form of plasmids, vectors,and transcription or expression cassettes, comprised of at least onepolynucleotide encoding a polypeptide of interest. Vectors are capableof directing the expression of genes to which they are operably linked.Such vectors are referred to herein as “recombinant expression vectors”or “expression vectors.” In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids. In thepresent specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most common vector form. However,the invention is intended to include other forms of expression vectorsthat serve equivalent functions, including, but not limited to, viralvectors (e.g., replication defective retroviruses, modifiedalphaviruses, adenoviruses and adeno-associated viruses).

Constructs that are suitable for expression of proteins in animal cellsare well known in the art. For example, polynucleotides may be operablylinked to an expression control sequence such as those present in thepMT2 or pED expression vectors disclosed in, e.g., Kaufman et al. (1991)Nuc. Acids Res. 19:4485-90. Other suitable expression control sequencesare found in vectors known in the art and include, but are not limitedto: HaloTag™ pHT2, pACT, pBIND, pCAT®3, pCI, phRG, phRL (Promega,Madison, Wis.); pcDNA3.1, pcDNA3.1-E, pcDNA4/HisMAX, pcDNA4/HisMAX-E,pcDNA3.1/Hygro, pcDNA3.1/Zeo, pZeoSV2, pRc/CMV2, pBudCE4 pRc/RSV(Invitrogen, Carlsbad, Calif.); pCMV-3Tag Vectors, pCMV-Script® Vector,pCMV-Tag Vectors, pSG5 Vectors (Stratagene, La Jolla, Calif.);pDNR-Dual, pDNR-CMV (Clonetech, Palo Alto, Calif.); and pSMEDA (Wyeth,Madison, Wis.). General methods of expressing recombinant proteins arealso known and are exemplified in, e.g., Kaufman (1990) Meth. Enzymol.185:537-66.

As defined herein “operably linked” means enzymatically or chemicallyligated to form a covalent bond between the polynucleotide to beexpressed and the expression control sequence in a manner that theencoded protein is expressed by the transfected host cell.

The recombinant expression constructs of the invention may carryadditional sequences, such as regulatory sequences (e.g., sequences thatregulate either vector replication (e.g., origins of replication,transcription of the nucleic acid sequence encoding the polypeptide (orpeptide) of interest) or expression of the encoded polypeptide), tagsequences such as histidine, and selectable marker genes. The term“regulatory sequence” is intended to include promoters, enhancers andany other expression control elements (e.g., polyadenylation signals,transcription splice sites) that control transcription, replication ortranslation. Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology, AcademicPress, San Diego, Calif. (1990). Those skilled in the art will recognizethat the design of the expression vector, including the selection ofregulatory sequences, will depend on various factors, including choiceof the host cell and the level of protein expression desired. Preferredregulatory sequences for expression of proteins in mammalian host cellsinclude viral elements that direct high levels of protein expression,such as promoters and/or enhancers derived from the FF-1a promoter andBGH poly A, cytomegalovirus (CMV) (e.g., the CMV promoter/enhancer),Simian virus 40 (SV40) (e.g., the SV40 promoter/enhancer), adenovirus(e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Viralregulatory elements, and sequences thereof, are described in, e.g., U.S.Pat. Nos. 5,168,062; 4,510,245; and 4,968,615, all of which areincorporated by reference herein in their entireties.

Suitable vectors, containing appropriate regulatory sequences, includingpromoter sequences, terminator sequences, polyadenylation sequences,enhancer sequences, marker genes and other sequences as appropriate, maybe either chosen or constructed. Inducible expression of proteins,achieved by using vectors with inducible promoter sequences, such astetracycline-inducible vectors, e.g., pTet-On™ and pTet-Off™ (Clontech,Palo Alto, Calif.), may also be used in the disclosed method. Forfurther details regarding expression vectors, see, for example,Molecular Cloning: a Laboratory Manual (2nd ed.) eds. Sambrook et al.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).Many known techniques and protocols for manipulation of nucleic acids,for example, in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells, gene expression, andanalysis of proteins, are also described in detail in Current Protocolsin Molecular Biology (2nd ed.) eds. Ausubel et al., Wiley & Sons,Alameda, Calif. (1992).

A polynucleotide inserted into an expression construct for producing apolypeptide of interest may encode any polypeptide that is capable ofbeing expressed in the host cell used in the cell culture. Thus, thepolynucleotide may encode full-length gene products, portions offull-length genes, peptides, or fusion proteins. Such polynucleotidesmay consist of genomic DNA or cDNA, and may be derived from any animal.Polynucleotides may be isolated from cells or organisms by methods wellknown in the art, e.g., PCR or RT-PCR, or may be produced by knownconventional chemical synthesis methods. Such chemically syntheticpolynucleotides may possess biological properties in common with naturalpolynucleotides, and thus may be employed as substitutes for naturalpolynucleotides.

Polypeptides may also be recombinantly produced by operably linking thepolynucleotide encoding the polypeptide of interest to suitable controlsequences in one or more insect expression vectors, such as baculovirusvectors, and employing an insect cell expression system. Materials andmethods for baculovirus/Sf9 expression systems are commerciallyavailable in kit form (e.g., the MAXBAC® kit, Invitrogen, Carlsbad,Calif.).

Transfection of host cells, e.g., animal cells, with the expressionconstruct may be achieved by numerous methods that are well known in theart. Cells may be either transiently transfected or stably transfected.Several different well-established methods exist for the delivery ofmolecules, particularly nucleic acids, into host cells, e.g., animalcells. Depending on the cell type, the desired transfection (i.e.,transient or stable), and the specific experimental requirements, suchas transfection of difficult cell lines or primary cells, the type ofmolecule transfected (genomic DNA, DNA, oligonucleotides), or theexpression construct chosen, each transfer method possesses advantagesand disadvantages known to those of skill in the art. Commontransfection methods include, e.g., calcium phosphate precipitation,liposome mediated transfection, DEAE Dextran-mediated transfection, geneguns, electroporation, nanoparticle delivery, polyamines, episomes, andpolyethylenimines. In addition, numerous transfection kits and reagentsare commercially available from companies such as Invitrogen (VOYAGER™,LIPOFECTIN®), EMD Biosciences, San Diego, Calif. (GENEJUICE™), Qiagen,Germantown, Md. (SUPERFECT™), Orbigen, San Diego, Calif. (SAPPHIRE™),and many others known to those of skill in the art. Transfectionprotocols may also be found in Basic Methods in Molecular Biology(2^(nd) ed.) eds. Davis et al., Appleton and Lange, CT (1994).

The present invention uses cell cultures, e.g., large-scale animal cellcultures, to produce large quantities of the polypeptide of interest.Methods for large-scale transient transfections are disclosed inLarge-scale Mammalian Cell Culture Technology (Biotechnology andBioprocessing Series) ed. Lubiniecki, Marcel Dekker, NY (1990);Kunaparaju et al. (2005) Biotechnol. Bioeng. 91:670-77; Maiorella et al.(1988) Bio/Technology 6:1406-10; Baldi et al., supra; Lan Pham et al.,supra; Meissner et al., supra; Durocher et al., supra). In general,large-scale transient gene expression in mammalian cell cultures mayemploy any one of several common types of transfection modes, e.g.,polyethylenimine, electric field pulse, CALFECTION™ or calciumphosphate, to achieve high transfection efficiency at desired scales orvolumes, e.g., greater that 10 liters (Derouazi et al., supra; Rols etal. (1992) Eur. J. Biochem. 206(1):115-21; Wunn and Bernard (1999) Curr.Opin. Biotechnol. 10(2):156-59; Schlaeger and Christensen (1999)Cytotechnology 30(1-3):71-83; Jordan et al. (1998) Cytotechnology26(1):39-47; Lindell et al. (2004) Biochim. Biophys. Acta1676(2):155-61). These large-scale cultures are generally grown inbioreactors, shakers, or incubators with stir plates, and may also beknown as “spinner” or “suspension” cultures. Thus, as opposed totraditional transfections, in which cells are attached to plates orflasks, the disclosed methods generally use suspension cultures.Large-scale cell cultures are generally considered to be cell culturesthat have a volume of greater than about 100 ml.

In some instances, cell lines expressing the polypeptide of interest maybe first produced and then used to seed a large-scale cell culture.Stable cell lines that express a protein of interest may be produced byvarious well-known methods, including the methods used for transienttransfection disclosed herein. In general, stable cell lines areproduced by long-term growth and selection in a chemically definedmedia. For example, cells transfected (e.g., by calcium phosphateprecipitation, or liposomal transfection) with a nucleic acid thatencodes a polypeptide of interest may concomitantly be transfected witha vector carrying a neomycin resistance gene, which confers resistanceto neomycin/geneticin (G418). The transfected cells are then grown inG418-containing media, and the surviving cells clonally expanded toproduce a stably expressing cell line. Aliquots of this cell line maythen be used to seed a large-scale culture and to produce largequantities of the protein of interest.

Transfecting cells requires the optimization of several variables,including cell-seeding density (e.g., about 1×10⁵ to about 3×10⁶cells/ml culture), serum concentration (e.g., 0-10%), incubationtemperature (e.g., about 20-38° C.), transfection vehicle or reagent(chemical or electric), culture volume (e.g., about 5 ml-20 liters), andincubation time (e.g., about 24-144 hours). For each cell type, optimalparameters will vary. However, commercial suppliers generally provideoptimization guidelines for transfecting particular cell types, as dovarious references known to those of skill in the art that utilizetransfection of the host cell chosen. These sources may be used todirect transfection of the chosen host cell, or may be used as astarting point from which simple trial and error may be used to provideoptimum transfection parameters.

Cell Culture

Typical procedures for producing a polypeptide of interest include batchcultures and fed-batch cultures. Batch culture processes traditionallycomprise inoculating a large-scale production culture with a seedculture of a particular cell density, growing the cells under conditionsconducive to cell growth and viability, harvesting the culture when thecells reach a specified cell density, and purifying the expressedpolypeptide. Fed-batch culture procedures include an additional step orsteps of supplementing the batch culture with nutrients and othercomponents that are consumed during the growth of the cells. One ofordinary skill in the art will recognize that the present invention canbe employed in any system in which cells are cultured including, but notlimited to, batch, fed-batch and perfusion systems. In certain preferredembodiments of the present invention, the cells are grown in fed-batchsystems.

A persistent and unsolved problem with traditional cultures, e.g., batchand fed-batch cultures, is the production of metabolic waste products,which have detrimental effects on cell growth, viability, and productionof expressed polypeptides. Two metabolic waste products that haveparticularly detrimental effects are lactate and ammonium, which areproduced as a result of glucose and glutamine metabolism, respectively.In addition to the enzymatic production of ammonium as a result ofglutamine metabolism, ammonium also accumulates in cell cultures as aresult of nonmetabolic degradation over time.

Traditional media formulations, including commercially available mediasuch as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM],Sigma), contain relatively high levels of glucose and glutamine (thelatter in comparison to other amino acids). Previously, these componentswere believed to be required in abundance since they are the primarymetabolic energy sources for the cells. However, rapid consumption ofthese nutrients leads to the accumulation of lactate and ammonium asdescribed above. Additionally, high initial levels of glucose andglutamine, and the subsequent accumulation of lactate and ammonium,result in high osmolarity, a condition that by itself is oftendetrimental to cell growth, cell viability and the production ofpolypeptides. The rationally designed medium disclosed herein may bemodified to decrease the accumulation of harmful metabolic products.Such modifications may be found in, e.g., U.S. Published PatentApplication Nos. 2005/0070013 (restricted glucose feeding) and2006/0121568 (modifications of amino acid content and ratios) (both ofwhich are hereby incorporated by reference herein in their entireties).

Rational Media Design and Formulations

Traditional media formulations begin with a relatively low level oftotal amino acids in comparison with the media formulations of thepresent invention. For example, DME-F12 (a 50:50 mixture of Dulbecco'sModified Eagle's medium and Ham's F12 medium) has a total amino acidcontent of 7.29 mM, and the traditional cell culture medium known asRPMI-1640 has a total amino acid content of 6.44 mM (see e.g., Morton(1970) In Vitro 6:89-108; Ham (1965) Proc. Nat. Acad. Sci. U.S.A.53:288-93; Moore et al. (1967) J. Am. Medical Assn. 199:519-24, allincorporated by reference herein). More recent media formulations (suchas the media disclosed in U.S. Published Patent Application No.2006/0121568) contain higher levels of amino acids and nutrients.Traditional formulations, however, are not based on actual calculatedcell requirements, which include cell growth, cell maintenance, and, forcell cultures used to produce recombinant polypeptides, productionrequirements. Using these variables, provided herein are methods ofdetermining media formulations with much higher, and yet nontoxic,concentrations of total amino acids.

The cell culture media formulations, e.g., the large-scale cell culturemedia formulations, described herein, when used in accordance with,e.g., other culturing steps described herein, and with, e.g.,modifications such as those found in, e.g., U.S. Published PatentApplication No. 2006/0121568, optimize cell density and polypeptidetiter. An amino acid concentration of the media formulations describedherein is based on the concentration of the amino acid(s) requiredfor: 1) cell mass; 2) cell maintenance; and 3) polypeptide production.In one embodiment of the invention, a cell culture medium contains aconcentration of the amino acid(s) that is calculated for use in cellmass, a concentration of the amino acid(s) that is calculated for use incell maintenance, and a concentration of the amino acid(s) that iscalculated for incorporation into the polypeptide of interest. Inanother embodiment of the invention, a cell culture medium contains aconcentration, A, of an amino acid that is represented by the formulaA=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is the concentration of the aminoacid that is used per unit of cell mass, P is the concentration of theamino acid that is used for incorporation into the polypeptide ofinterest per unit of polypeptide titer, M is the multiplier for thedesired cell mass (i.e., the desired peak units of cell mass), N is themultiplier for the desired concentration of the polypeptide of interest(i.e., desired or target polypeptide titer), Y is the cell maintenancefactor; and F is the baseline factor.

The concentration, P, of the amino acid that is used for incorporationinto the polypeptide of interest per unit of polypeptide titer in theformula above is based on the primary structure of the recombinantprotein, i.e., the amino acid content of the polypeptide. Thus, P willvary based on the polypeptide of interest that is to be produced by thelarge-scale cell culture. P may then be converted to the amino acidrequirement for the target concentration of the polypeptide of interestusing N, the multiplier for the desired concentration of the polypeptideof interest (i.e., desired or target polypeptide titer). In somerepresentative examples of the invention below, the basic unit ofpolypeptide titer is 1 g/L. In Table 1, below, which contains arepresentative calculation using the formula supplied herein, the aminoacid concentration of the cell culture medium that is required for thepolypeptide of interest at a titer of 10 g/L (column 4) is determined bymultiplying the concentration, P, of the amino acid required for 1 g/L(column 2) by the multiplier, N, wherein N=10.

TABLE 1 Representative Determination of Baseline-Adjusted Amino AcidConcentration Required For Target Titer of 10 g/L Antibody at a DesiredCell Mass, Where Desired Cell Mass Is Represented by Desired Peak CellDensity of 15 × 10⁶ cells/ml 7 8 (A) (Column 7 × F) Calculated Baseline-3 (X) 4 (P × N) 5 (X × M) 6 (X × M × Y) Total AA Adjusted AA AA Total AATotal AA Total AA Concentration Concentration ConcentrationConcentration Concentration Concentration Required for Required for 2(P) Required for Required for Required for Required for Target Target AACell Mass Target Desired Peak Cell Antibody Titer Antibody TiterConcentration Represented Antibody Titer Cell Density of Maintenance andDesired and Desired Required For by Cell of 10 g/L 15 × 10⁶ cells/ml (Y= 100%) or Peak Cell Peak Cell 1 Initial Density (N = 10) (M = 15) (Y= 1) Density Density Amino Antibody of 10⁶ Column Column Column 5 ×(Column (F = 1.3) Acid Titer of 1 g/L cells/ml 2 × 10 3 × 15 100% 4 +5 + 6) Column 7 × 1.3 (AA) mM mM mM mM mM mM mM ALA 0.41 0.30 4.08 4.564.56 13.20 17.17 ARG 0.17 0.16 1.73 2.38 2.38 6.50 8.45 ASN 0.26 0.152.62 2.23 2.23 7.08 9.21 ASP 0.29 0.29 2.87 4.36 4.36 11.59 15.07 CYS0.19 0.09 1.88 1.42 1.42 4.73 6.14 GLU 0.40 0.16 4.00 2.33 2.33 8.6711.26 GLN 0.39 0.29 3.87 4.40 4.40 12.67 16.47 GLY 0.47 0.25 4.72 3.803.80 12.33 16.02 HIS 0.16 0.07 1.59 1.07 1.07 3.72 4.83 ILE 0.14 0.161.43 2.33 2.33 6.10 7.93 LEU 0.49 0.25 4.88 3.80 3.80 12.49 16.24 LYS0.52 0.24 5.20 3.55 3.55 12.31 16.00 MET 0.10 0.06 0.97 0.86 0.86 2.693.50 PHE 0.23 0.12 2.34 1.78 1.78 5.89 7.65 PRO 0.59 0.16 5.94 2.33 2.3310.61 13.79 SER 0.97 0.34 9.73 5.11 5.11 19.96 25.94 THR 0.68 0.20 6.803.04 3.04 12.88 16.75 TRP 0.16 0.04 1.64 0.56 0.56 2.75 3.58 TYR 0.350.12 3.48 1.78 1.78 7.03 9.14 VAL 0.74 0.23 7.36 3.50 3.50 14.36 18.67Total 7.71 3.68 77.13 55.21 55.21 187.55 243.82 (mM)

The multiplier N may be calculated, e.g., by multiplying the integratedviable cell density (IVC) by the specific productivity (qp) of aparticular cell line (N=IVC*qp). For example, if the seed density of aparticular cell line is 0.8×10⁶ cells/ml, the cell density at day 6 andday 10 is 15×10⁶ cells/ml, and the cell density at day 18 is 11×10⁶cells/ml (i.e., 73% of the value at days 6 and 15), then the IVC=211×10⁶(cells/ml)*day (i.e., [(0.8+15)/2]*6 days+[(15+15)/2]*4days+[(15+11)/2]*8 days). If the average specific productivity (qp) ofthe chosen cell line is, e.g., 47 μg/10⁶ cells/day, then N=10 g/L at day18 (i.e., 211×10⁶ (cells/ml)*day×47 μg/10⁶ cells/day). One of skill inthe art will realize that these calculations may be performed with anycell line, or that N may be estimated based on cell characteristics andorigin. Alternatively, the multiplier N need not be calculated from IVCand qp, and may simply be a reasonable target titer for a particularcell culture. A prophetic example, describing the calculation of IVC andqp, and the further selection of reasonable N and M values, is providedas Example 5 (below).

As used herein, “cell mass,” “cell density,” and the like refer to acollection of cells. For example, a cell mass can refer to a cellpellet. As used herein, “desired cell mass” and the like refers to acollection of cells, e.g., a cell pellet, that a practitioner desires toobtain in a cell culture. As used herein, “unit of cell mass” and thelike reflects a number of ways of representing cell mass, e.g., cellnumber, cell density, cell volume, packed cell volume, dry cell weight,etc. One skilled in the art will know which is the most convenient orappropriate way of representing a unit of cell mass, etc., for aparticular experimental condition. One skilled in the art will alsounderstand that, depending on the unit of cell mass, M, the multiplierfor desired cell mass, i.e., the desired peak units of cell mass, willbe represented by either desired peak cell numbers, desired peak celldensity, desired peak cell volume, desired peak packed cell volume,desired peak dry weight, etc.

In one embodiment of the invention, the unit of cell mass is representedby cell density and the desired cell mass is represented by desired peakcell density. In another embodiment, the unit of cell mass isrepresented by dry cell weight or mass. The dehydrated cell massconsists essentially of all proteins, carbohydrates, lipids, and nucleicacids present in that cell mass. Thus, the concentration, X, of an aminoacid can be determined experimentally, by first spinning a known numberof cells to a cell pellet, drying the cell pellet, and subsequentlyexposing the dried pellet to acid-hydrolysis, thereby lysing thecellular proteins of the cell pellet to individual amino acids, whichmay then be quantified by an amino acid analyzer (see, e.g., Example 1).This provides the amino acid concentration, X, of a given number ofcells, which may then be converted to the amino acid requirement for thedesired peak cell density using M, the multiplier for the desired peakcell density. In Table 1, the total amino acid concentration of the cellculture medium that is required for the desired peak cell density of15×10⁶ cells/ml (column 5) is determined by multiplying theconcentration, X, of the amino acid required for 10⁶ cells/ml (column 3)by the multiplier, M, wherein M=15. Thus, in some representativeexamples of the invention, the unit of cell mass is 10⁶ cells/ml.Alternatively, e.g., for an amino acid known to be susceptible todegradation, the concentration, X, of the amino acid that is used in theformula above may be determined from literature values, e.g., Nyberg etal. (1999) Biotechnol. Bioeng. 62:324-35; Nadeau et al. (2000) Metab.Eng. 2:277-92; and Bonarius (1996) Biotechnol. Bioeng. 50:299-318, orthe methods disclosed in such publications or similar publications knownin the art.

When the multiplier M for desired cell mass is represented desired peakcell density, M may be chosen as, e.g., the peak density of a particularcell line, by the density at which the productivity of the cell line ismaximized, or by the predicted density for the cell line at a particulartime period based on the specific growth rate.

The amino acid concentration of the cell culture medium that is requiredfor cell maintenance, Y, is a percentage of the amino acid concentrationrequired for the desired cell mass, e.g., desired peak cell density. Inone embodiment of the invention, the maintenance requirement ranges from0% to 300% of the desired cell mass requirement, which providessufficient nutrients for cell use without risk of nutrient-inducedtoxicity. In another embodiment of the invention, the maintenancerequirement ranges from 0% to 150% of the desired cell mass requirement,which provides sufficient nutrients for cell use without risk ofnutrient-induced toxicity. The maintenance requirement increases asculture duration increases (e.g., 100-150% maintenance for a 21 dayculture). In Table 1, the amino acid concentration of the cell culturemedium that is required for cell maintenance (column 6) is determined bymultiplying the amino acid concentration required for the desired peakcell density (column 5) by the cell maintenance factor, Y, which in thisrepresentative example is 100%, in order to allow an extended cultureperiod. The maintenance factor, Y, will differ for different cells (anddifferent cell lines) depending on the unique metabolic demands of thecells in culture. Further, the maintenance requirements for cells inculture will also differ due to the variability in processes, e.g.,inoculation density, culture duration, time of temperature shift, etc.As an initial guideline, one may provide, e.g., 0% maintenance (daily)for cultures at days 0 to 5, 3% to 5% maintenance (daily) for culturesat days 6 to 10, and 7% to 10% maintenance (daily) for cultures at days11 to 21. For a process greater than 21 days, cultures may be provided2% to 5% maintenance (daily) for those additional days. One of ordinaryskill in the art will realize that adjusting the maintenance factor, Y,to optimize density and titer, is merely a matter of routine trial anderror. Adjusting amino acid concentration according to the cellmaintenance requirement is important for increased viability andproductivity of the cell culture, and thus is an important aspect of thepresent invention. For example, adjusting amino acid concentrationaccording to the cell maintenance requirement enables the cell cultureto sustain greater cell density, cell viability, and to produce higherpolypeptide titer (see, e.g., Example 6).

Once the amino acid requirements of desired: 1) cell mass (column 5); 2)cell maintenance (column 6); and 3) polypeptide production for thetarget titer (column 4) are determined, the calculated total amino acidconcentration of the target cell culture may be obtained. In Table 1,the calculated total amino acid concentration of the cell culture mediumthat is required for the target cell culture (column 7), is determinedby adding the amino acid concentration of the cell culture medium thatis required for the polypeptide of interest at a titer of 10 g/L (column4), the amino acid concentration of the cell culture medium that isrequired for the desired peak cell density of 15×10⁶ cells/ml (column5), and the amino acid concentration of the cell culture medium that isrequired for a selected level of cell maintenance (column 6).

Once the calculated total amino acid concentration of the cell culturemedium that is required for the target cell culture is obtained asdescribed above, the value is adjusted to a desired cell culture mediumamino acid concentration, A, by a baseline factor, F, which allows forthe driving force of amino transfer, e.g., the extra amino acidsrequired for mass transfer, the extra amino acids required to drivetransport across cell membrane, etc. This adjusted value, A, is referredto herein as the “baseline-adjusted amino acid concentration” or“optimized concentration.” The baseline-adjusted amino acidconcentration, A, represents the cumulative total amount of an aminoacid(s) that will be delivered to the culture, expressed relative to thefinal volume of the culture, which includes the volume of the startingmedium, plus the volume of any feeds for perfusion or fed-batchculture(s). Adjusting the total amino acid concentration to thebaseline-adjusted amino acid concentration is an important aspect of theinvention because it allows higher cell viability, cell density, andpolypeptide titers (see, e.g., Example 6).

The baseline factor, F, which increases the calculated total amino acidconcentration of the cell culture medium by up to 200%, ranges from 1(0% increase) to 3 (200% increase). In one embodiment of the invention,the range for F is between 1 and 1.5. In another embodiment of theinvention, a value of F below 1 may be offset by modifying thecalculated total amino acid concentration of the cell culture medium(Table 1, column 7), which may be achieved by modifying the amino acidconcentration required for desired cell mass, the amino acidconcentration required for cell maintenance, and/or the amino acidconcentration required for incorporation into the polypeptide ofinterest. For example, a baseline factor of 0.5 may be offset byincreasing the calculated total amino acid concentration of the cellculture medium by, e.g., a factor of two (or more), which may beachieved by varying M, X, N, P and/or Y. In Table 1, thebaseline-adjusted amino acid concentration, A, of the cell culturemedium that is required for the target titer and desired peak celldensity (column 8), is determined by multiplying the calculated totalamino acid concentration of the cell culture medium (column 7), by thebaseline factor, F, which in the representative example of Table 1 is1.3 (corresponding to a 30% increase over the calculated total aminoacid concentration of the cell culture medium).

Medium containing the baseline-adjusted concentration, A, of an aminoacid is referred to herein as the “desired cell culture medium.” Thus,the “desired cell culture medium” represents a goal medium that containsthe baseline-adjusted concentration, A. This medium comprises at leastone amino acid concentration determined by the above formula. Preferablythe desired cell culture medium contains more than one amino acidconcentration determined by the above formula. More preferably, thedesired cell culture medium contains at least twelve adjusted amino acidconcentrations, e.g., an adjusted concentration of arginine, histidine,isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine, determined by the above formula. Itwill be understood by one of skill in the art that the baseline-adjustedamino acid concentration, A, in the desired cell culture medium may beachieved by any number of means, including, but not limited to,individually adding the amino acid(s), adding peptone or other proteinhydrolysates, and/or by adding another concentrated cell culture medium(e.g., a feeding cell culture medium (or medium mix, e.g., a mediumpowder)) to a starting cell culture medium (or starting cell culturemedium mix, e.g., a medium powder). One of skill in the art willunderstand that the addition of peptone (or other protein hydrolysate)may be directed by the amino acid contents of the particular peptoneproduct of choice, or by determining the amino acid concentrationprovided by a particular peptone, e.g., in general, 5 g/L peptoneprovides a concentration of about 40 mM to about 50 mM total aminoacids.

The amino acid concentration in the desired cell culture medium is basedon at least one baseline-adjusted amino acid concentration, A,determined by the inventive formula disclosed herein; however, thatconcentration, as well as other concentrations of amino acids in thedesired cell culture medium, may be varied from the baseline-adjustedamino acid concentration(s) due to the influence of several factors. Forexample, certain amino acids may be produced during culturing, and thusmay be kept at a low level. Other amino acids may be varied based onpublished values (see, e.g., U.S. Published Patent Application No.2006/0121568). Further, some amino acids, e.g., methionine, may beconsumed at a greater rate by particular cell types, and thus should beadded in excess. Yet other amino acids, such as proline, provide adriving force for cell growth and polypeptide production (see Example4), and these amino acids should be provided, in some cases, at agreater amount than determined by the above inventive formula. Inaddition, one may modify the baseline-adjusted amino acid concentrationif the concentration obtained using the above formula is considered tobe toxic (e.g., consideration of the levels of serine, tyrosine,methionine and valine). It is within the knowledge of one of skill inthe art, upon obtaining the baseline-adjusted amino acid concentration,A, of an amino acid(s) for use in the desired cell culture medium, tovary the baseline-adjusted amino acid concentration based on factorssuch as those noted herein.

Additional media components, for example, vitamins, salts, glucose,elements, may be calculated from (or based on) various sources, e.g.,U.S. Published Patent Application Nos. 2005/0070013 and 2006/0121568.Further, the baseline-adjusted amino acid concentration, A, of an aminoacid obtained using the above formula may be modified to provide aparticular ratio in relation to another amino acid (e.g., the ratio ofglutamine to asparagine) or to fall within a desired combinedconcentration (e.g., the combined concentration of glutamine andasparagine). For example, it is known that a high asparagine, lowglutamine medium, combined with temperature shift, enables uptake oflactate, thereby detoxifying a cell culture (U.S. Published PatentApplication No. 2006/0121568). Thus, one may wish to modify thebaseline-adjusted concentration, A, of glutamine and/or asparagine, inorder to obtain an optimum ratio.

It will be noted from the representative example in Table 1 that thecombined concentration of the baseline-adjusted amino acidconcentrations for use in the desired cell culture medium is high, i.e.,over 243 mM. Thus, disclosed herein is the finding that a highconcentration of amino acids may be used in a desired cell culturemedium without toxicity or titer detriment if that concentration isbased upon the calculated amino acid requirements for a target celldensity and polypeptide titer. In one embodiment of the invention, thecombined concentration of amino acids in the desired cell culture mediumis between about 120 mM and about 250 mM. In other embodiments, thecombined concentration of amino acids in the desired cell culture mediumis greater than about 250 mM, e.g., about 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500 or 510 mM, or any intermediate value.

The above-identified baseline-adjusted concentration, A, of an aminoacid that is used in a desired cell culture medium, may be used inbatch, fed-batch, and perfusion cultures. When used in batch culture,the initial concentration of the amino acid used in the desired cellculture medium is the baseline-adjusted amino acid concentration, A.When used in fed-batch or perfusion cultures, the baseline-adjustedamino acid concentration, A, represents the cumulative total amount ofan amino acid(s) that will be delivered to the culture, which includesthe volume of the starting medium plus the volume of all feeds. Thus,for fed-batch cultures, which use either continuous feeds (e.g., feedson days 3-21) or periodic feeds (e.g., feeds every 2-3 days), thestarting medium is engineered to contain a starting concentration of theamino acid, B, according to the formula B=[A−(Z*V)]/(1−V), wherein Z isthe concentration of the amino acid in the feeding cell culture medium,and V is the volume of the feeding culture medium as a proportion of thedesired cell culture medium volume. A representative example of thecalculations required to obtain the starting amino acid concentration,B, is provided in Table 2, below. In Table 2, some baseline-adjustedamino acid concentrations, A (column 2), are converted to the startingmedia amino acid concentration, B (column 5), based on a 17% feed volume(V=17%), and the feeding medium amino acid concentration, Z (column 4).In this example, several baseline-adjusted amino acid concentrations, A(column 2), and starting amino acid concentrations, B (column 5), aremodified to the values shown in bold in columns 3 and 6. Modification ofasparagine, aspartic acid, glutamine, and cysteine concentrations wasbased on the concentrations suggested by U.S. Published PatentApplication No. 2006/0121568; methionine was adjusted by 50% tocompensate for its consumption at a higher amount than predicted;alanine, glutamic acid and glycine are produced by the cultures (andthus kept at a low level); and serine, tyrosine, and valineconcentrations were decreased to nontoxic levels. It will therefore beunderstood that the starting amino acid concentration, B, may be basedon either the baseline-adjusted amino acid concentration, A, or on themodified baseline-adjusted amino acid concentration.

One of skill in the art will appreciate that the feeding medium used toobtain the desired cell culture medium during fed-batch and perfusioncell culturing should be as highly concentrated as possible in order toavoid overflow in the container in which the culture is carried out(e.g., a bioreactor or shaker flask) and to avoid diluting the mediacomponents. In the example set forth in Table 2, a preferred feedingmedia is denoted “Feed Medium,” and the amino acid concentrations, Z, ofthe Feed Medium are set forth in column 4. However, any highlyconcentrated feeding medium, or any method of providing highlyconcentrated amino acids to the starting cell culture medium may beused, as long as the desired baseline-adjusted amino acid concentration,A, will be achieved in the target volume. Such methods of providinghighly concentrated amino acids to a cell culture are commonly used andwell known to one of skill in the art.

It will be understood by one of skill in the art that the startingconcentration of the amino acid, B, in the starting cell culture mediummay be achieved by any number of means, including, but not limited to,individually adding the amino acid(s), adding peptone and/or otherprotein hydrolysate, and/or adding another concentrated culture medium(or medium mix, e.g., a medium powder) to the starting cell culturemedium (or starting cell culture medium mix, e.g., a medium powder). Itwill also be understood by one of skill in the art that theconcentration of the amino acid, Z, in the feeding cell culture mediummay be achieved by any number of means, including, but not limited to,individually adding the amino acid(s), adding peptone and/or otherprotein hydrolysate, and/or adding another concentrated culture medium(or medium mix, e.g., a medium powder) to the feeding cell culturemedium (or feeding cell culture medium mix, e.g., a medium powder).

It will be noted from the representative example in Table 2 that thecombined concentration of amino acids for use in the starting cellculture medium is high, i.e., over 125 mM. Thus, disclosed herein is thefinding that a high concentration of amino acids may be used withouttoxicity or titer detriment in a starting cell culture medium if thestarting amino acid concentration is based upon the calculated aminoacid requirements for a desired peak cell density and a desiredpolypeptide titer. In one embodiment of the invention, the combinedconcentration of amino acids is between about 70 mM and about 510 mM. Inone embodiment of the invention, the combined concentration of aminoacids is between about 120 mM and about 350 mM. In another embodiment ofthe invention, the combined concentration of amino acids in the startingcell culture medium is between about 70 mM and about 140 mM. In anotherembodiment of the invention, the combined concentration of amino acidsin the starting cell culture medium is greater than about 140 mM.

TABLE 2 Representative Determination of Starting Amino AcidConcentration For A Target Titer of 10 g/L Antibody, a Desired Peak CellDensity of 15 × 10⁶ cells/ml, and a 17% Feed 1 2 (A) 3 4 (Z) 5 (B) 6Modified Baseline- Starting AA Starting AA Adjusted AA Modification ofConcentration Concentration Concentration Baseline-Adjusted Required ForRequired for Required For AA Concentration Target Titer and Target TiterTarget Titer and Required for Target Feed Desired Peak and Desired AminoDesired Peak Titer and Desired Medium AA Cell Density Peak Cell Acid(AA) Cell Density Peak Cell Density Concentration (V = 17%) Density mMmM mM mM mM ALA 17.17 0.20 6.4 −1.07 0.2 ARG 8.45 8.45 35.13 2.99 2.99ASN 9.21 24.00 56 17.45 17.45 ASP 15.07 1.70 16 −1.23 1.7 CYS 6.14 0.400 0.48 0.4 GLU 11.26 0.20 6.4 −1.07 0.2 GLN 16.47 4.00 0 4.82 4.2 GLY16.02 4.00 6.4 3.51 3.51 HIS 4.83 4.83 11.2 3.53 3.53 ILE 7.93 7.9328.82 3.65 3.65 LEU 16.24 16.24 41.53 11.06 11.06 LYS 16.00 16.00 3212.72 12.72 MET 3.50 5.25 12.8 3.71 3.71 PHE 7.65 7.65 16 5.94 5.94 PRO13.79 13.79 19.2 12.68 12.68 SER 25.94 25.94 48.15 21.39 10.2 THR 16.7516.75 25.6 14.94 14.94 TRP 3.58 3.58 5.11 3.27 3.27 TYR 9.14 9.14 12.758.40 5.2 VAL 18.67 18.67 25.6 17.25 10.2 Total 243.82 405.09 127.73 (mM)

As shown in Table 3A and Table 3B, the determination of thebaseline-adjusted amino acid concentration of an amino acid, A, used ina desired cell culture medium, and the determination of the startingamino acid concentration, B, used in the starting cell culture medium,may be calculated for any desired target polypeptide titer and desiredpeak (target) cell density. The desired peak cell density of thelarge-scale culture ranges from about 3 to about 40×10⁶ cells/mL forfed-batch culture. In one embodiment of the invention, the desired peakcell density ranges from about 5 to about 20×10⁶ cells/mL. The targettiter of the large-scale culture ranges from about 3 to about 25 g/L. Inyet another embodiment of the invention, the target titer of thelarge-scale culture ranges from about 5 to about 20 g/L. For example, inTable 3, desired peak cell density ranges from 10 to 15×10⁶ cells/mL,and target titer varies from 3 to 10 g/L.

TABLE 3A Representative Examples of Baseline-Adjusted Amino AcidConcentrations, A, for Various Target Titers (As Represented by aMultiplier for Target Polypeptide Titer, N) and Desired Peak CellDensities (As Represented by a Multiplier for Desired Peak Cell Density,M). A N = 10 N = 5 N = 3 M = 15 M = 12.5 M = 10 Amino Acid ConcentrationmM mM mM ALA 17.2 12.5 9.50 ARG 8.5 6.3 4.81 ASN 9.2 6.5 4.89 ASP 15.111.3 8.68 CYS 6.1 4.3 3.20 GLU 11.3 7.7 5.60 GLN 16.5 12.1 9.14 GLY 16.011.3 8.43 HIS 4.8 3.3 2.46 ILE 7.9 6.0 4.60 LEU 16.2 11.4 8.50 LYS 16.011.1 8.18 MET 3.5 2.5 1.87 PHE 7.7 5.4 3.99 PRO 13.8 8.9 6.36 SER 25.917.4 12.66 THR 16.8 11.0 7.93 TRP 3.6 2.3 1.61 TYR 9.1 6.1 4.43 VAL 18.712.4 8.94 Total 243.8 169.8 125.8

TABLE 3B Representative Examples of Starting Media Amino AcidConcentrations, B, for Various Target Titers (As Represented by aMultiplier for Target Polypeptide Titer, N) and Desired Peak CellDensities (As Represented by a Multiplier for Desired Peak Cell Density,M); Starting Media Amino Acid Concentrations Were Determined bySubtracting Feed and Other Modifications from Baseline-Adjusted AminoAcid Concentrations B N = 10 N = 5 N = 3 M = 15 M = 12.5 M = 10 AminoAcid Concentration mM mM mM ALA 0.2 0.2 0.20 ARG 3.0 1.9 1.68 ASN 17.414.6 10.77 ASP 1.7 1.7 1.70 CYS 0.4 0.4 0.40 GLU 0.2 0.2 0.20 GLN 4.04.0 4.00 GLY 3.5 3.6 3.75 HIS 3.5 2.2 1.56 ILE 3.6 2.5 2.10 LEU 11.1 6.95.09 LYS 12.7 7.9 5.73 MET 3.7 2.4 1.78 PHE 5.9 3.8 2.75 PRO 12.7 7.45.04 SER 10.2 10.2 9.00 THR 14.9 8.8 6.10 TRP 3.3 1.8 1.24 TYR 5.2 5.13.58 VAL 10.2 10.4 7.22 Total 127.5 95.9 73.89

Using the formula to determine baseline-adjusted amino acidconcentration(s) and to develop desired cell culture media formulationsfor large-scale polypeptide production, the inventors have identifiedseveral criteria that result in high titer and high cell densitycultures. The criteria for producing a titer higher than 5 g/L, whichare represented by the values shown in Table 4, include, but are notlimited to, one or more of the following: greater than or equal to about3 mM tyrosine; between about 7 mM and about 30 mM proline; between about7 mM and about 30 mM valine; between about 7 mM and about 30 mM leucine;between about 7 mM and about 30 mM threonine; between about 7 mM andabout 30 mM lysine; a combined concentration of leucine, lysine,proline, threonine and valine that is between about 35 mM and about 150mM; a combined concentration of leucine, lysine, threonine and valinethat is between about 60% to about 80% of the total essential aminoacids in the desired cell culture medium; a combined concentration ofthe essential amino acids in the desired cell culture medium that isbetween about 30% to about 50% of the total amino acids in the desiredcell culture medium; and/or a combined concentration of total aminoacids between about 75 mM and about 510 mM.

TABLE 4 Representative Examples of the Amino Acid Content andRelationships For Rationally Designed Media for Various Target Titersand Desired Peak Cell Densities (as noted above, the basic unit ofpolypeptide titer is 1 g/L, and the basic unit of cell mass is 10⁶cells/ml). N = 5 N = 10 N = 15 N = 15 N = 15 M = 10 M = 10 M = 10 M = 20M = 30 Y = 0 Y = 1 Y = 1 Y = 1 Y = 1.5 Amino F = 1 F = 1.3 F = 1.3 F =1.3 F = 1.3 Acid (mM) (mM) (mM) (mM) (mM) ALA 5.08 13.21 15.86 23.7737.62 ARG 2.46 6.39 7.51 11.65 18.88 ASN 2.80 7.28 8.98 12.85 19.62 ASP4.34 11.29 13.15 20.71 33.94 CYS 1.89 4.91 6.14 8.60 12.91 GLU 3.55 9.2411.84 15.89 22.96 GLN 4.87 12.66 15.18 22.80 36.15 GLY 4.90 12.73 15.7922.39 33.93 HIS 1.50 3.91 4.94 6.78 10.01 ILE 2.27 5.91 6.84 10.88 17.96LEU 4.98 12.94 16.12 22.71 34.25 LYS 4.97 12.92 16.30 22.46 33.23 MET1.06 2.76 3.39 4.88 7.50 PHE 2.35 6.11 7.63 10.71 16.09 PRO 4.53 11.7715.63 19.67 26.75 SER 8.27 21.51 27.83 36.70 52.21 THR 5.43 14.11 18.5323.81 33.04 TRP 1.19 3.10 4.16 5.13 6.82 TYR 2.92 7.60 9.87 12.94 18.33VAL 6.01 15.63 20.42 26.48 37.10 Total 75.37 195.97 246.11 341.81 509.28Concentration Total Essential 32.22 83.78 105.84 145.49 214.87 TotalBold 21.39 55.61 71.37 95.46 137.61 Bold/Essential 66% 66% 67% 66% 64%Bold/Total 28% 28% 29% 28% 27% Essential/Total 43% 43% 43% 43% 42%

In Table 4, the bold amino acids are valine, threonine, leucine andlysine, the italicized amino acids are the essential amino acids (e.g.,arginine, histidine, leucine, isoleucine, lysine, methionine,tryptophan, threonine, and valine; and for CHO cell cultures, proline isadditionally an essential amino acid; one of skill in the art is awareof variations in the definition of essential amino acids as it appliesto different cells, etc.). Table 4 provides representative desired cellculture media for CHO cells at a variety of target cell densities (M),target titers (N), cell maintenance requirements (Y), and baselineadjustments (F).

One of skill in the art will recognize that the rationally designedmedia of the invention may be used to either produce a polypeptide ofinterest, or may be used in cell culturing that is not designed forpolypeptide production. Accordingly, a rationally designed media may beused in the disclosed methods of cell culturing, e.g., for the efficientgrowth, replication and/or maintenance of cell cultures, e.g.,large-scale cell cultures, that do not contain host cells engineered toproduce an exogenous polypeptide of interest, or which are not culturedto produce an endogenous polypeptide of interest. In such an instance,the desired cell culture medium need not account for the amino acid(s)required for incorporation into the polypeptide of interest, and insteadcontains an amino acid concentration(s) based on the concentration ofthe amino acid(s) required for: 1) desired cell mass, and 2) cellmaintenance. Such a desired cell culture medium used in the disclosedmethods of cell culturing contains a baseline-adjusted amino acidconcentration, A′, according to the formula A′=[(M*X)+(Y*M*X)]*F,wherein X is a concentration of the amino acid that is used per unit ofcell mass, M is a multiplier for a desired peak cell mass (e.g., desiredpeak cell density, etc.) of the cell culture, Y is a cell maintenancefactor, and F is a baseline factor. A desired cell culture mediumproduced according to this formula is then provided to a cell cultureunder conditions that allow growth and replication of the cells in thecell culture. In one embodiment of the invention, the method of cellculturing uses a large-scale cell culture. In another embodiment of theinvention, the method of cell culturing uses animal cells.

Proline Addition to Cell Culture Media

Using the rational media design methods disclosed herein, it has beendetermined that maintaining high proline levels throughout the cultureperiod of cell culture, e.g., large-scale cell culture, results inincreased polypeptide titer and increased cell density. This proline“threshold” ranges from about 1 mM to about 2 mM, and a level of prolinein the cell culture maintained above this threshold appears to be adriving force for producing high cell density, high titer large-scalecell cultures. Interestingly, the proline-driven increased polypeptidetiter and cell viability concomitantly increases the culture'srequirement for additional amino acids (in order to satisfy theincreased consumption rate), which at least partially explains why therationally designed media formulations herein contain high amino acidconcentrations.

Providing a Cell Culture

Various methods of preparing mammalian cells for production ofpolypeptides by batch and fed-batch culture are well known in the art.As described above, a nucleic acid sufficient to achieve expression(typically a vector containing the nucleic acid encoding the polypeptideof interest and any operably linked genetic control elements) may beintroduced into the host cell line by any number of well-knowntechniques. Typically, cells are screened to determine which of the hostcells have actually taken up the vector and express the polypeptide orprotein of interest. Traditional methods of detecting a particularpolypeptide or protein of interest expressed by mammalian cells include,but are not limited to, immunohistochemistry, immunoprecipitation, flowcytometry, immunofluorescence microscopy, SDS-PAGE, Western blots,enzyme-linked immunosorbent assay (ELISA), high performance liquidchromatography (HPLC) techniques, biological activity assays andaffinity chromatography. One of ordinary skill in the art will be awareof other appropriate techniques for detecting expressed polypeptides orproteins. If multiple host cells express the polypeptide or protein ofinterest, some or all of the listed techniques can be used to determinewhich of the cells expresses that polypeptide or protein at the highestlevels.

Once a cell that expresses the polypeptide or protein of interest hasbeen identified, the cell is propagated in culture by any of the varietyof methods well known to one of ordinary skill in the art. The cellexpressing the polypeptide or protein of interest is typicallypropagated by growing it at a temperature and in a medium that isconducive to the survival, growth and viability of the cell. The initialculture can be of any volume, but is often of lower volume than theculture volume of the production bioreactor used in the final productionof the polypeptide or protein of interest, and frequently cells arepassaged several times in bioreactors of increasing volume prior toseeding the production bioreactor. The cell culture can be agitated orshaken to increase oxygenation of the medium and dispersion of nutrientsto the cells. Alternatively or additionally, special sparging devicesthat are well known in the art can be used to increase and controloxygenation of the culture. In accordance with the present invention,one of ordinary skill in the art will understand that it can bebeneficial to control or regulate certain internal conditions of a cellculture, including but not limited to pH (e.g., 6.6 to 7.6), temperature(e.g., 25° C. to 42° C.), levels of oxygen and carbon dioxide (e.g., O₂:10% to 80% and CO₂: 7% to 15%, throughout culture), and osmolality(e.g., a starting osmolality of 260 to 340 mOsm/kg), etc.

As used herein, the teem “inoculum” is used to refer to a volume ofcells containing the nucleic acid that expresses the polypeptide ofinterest, which is used to seed the production vessel in which thelarge-scale animal culture will occur, e.g., the production bioreactor.In one embodiment of the invention, the inoculum volume is about 60 to80% of the final volume.

The starting cell density in the production bioreactor can be chosen byone of ordinary skill in the art. In accordance with the presentinvention, the starting cell density in the production bioreactor can beas low as a single cell per culture volume. In preferred embodiments ofthe present invention, starting cell densities in the productionbioreactor can range from about 0.1×10⁶ viable cells per mL to about10×10⁶ viable cells per mL and higher.

Initial and intermediate cell cultures may be grown to any desireddensity before seeding the next intermediate or final productionbioreactor. In one embodiment of the invention, the inoculum celldensity is about 0.5−1×10⁶ cells/ml. It is preferred that most of thecells remain alive prior to seeding, although total or near totalviability is not required. In one embodiment of the present invention,the cells may be removed from the supernatant, for example, by low-speedcentrifugation. It may also be desirable to wash the removed cells witha medium before seeding the next bioreactor to remove any unwantedmetabolic waste products or medium components. The medium may be themedium in which the cells were previously grown or it may be a differentmedium or a washing solution selected by the practitioner of the presentinvention.

The cells may then be diluted to an appropriate density for seeding theproduction bioreactor. The cells can be diluted into another medium orsolution, e.g., the starting cell culture medium or the desired cellculture medium, depending on the needs and desires of the practitionerof the present invention or to accommodate particular requirements ofthe cells themselves (e.g., if the cells are to be stored for a shortperiod of time prior to seeding the production bioreactor).

Initial Growth Phase

Once the production vessel is seeded as described above, the animal cellculture may be maintained in the initial growth phase using the desiredcell culture medium obtained by the inventive formula disclosed herein,and under conditions conducive to the survival, growth and viability ofthe cell culture. The precise conditions will vary depending on the celltype, the organism from which the cell was derived, and the nature andcharacter of the expressed polypeptide or protein.

A production bioreactor can be any volume that is appropriate forlarge-scale production of polypeptides or proteins. In a preferredembodiment, the volume of the production bioreactor is at least 500liters. In other preferred embodiments, the volume of the productionbioreactor is 1000, 2500, 5000, 8000, 10,000, 12,000 liters or more, orany intermediate volume. One of ordinary skill in the art will be awareof, and will be able to choose, a suitable bioreactor for use inpracticing the present invention. The production bioreactor may beconstructed of any material that is conducive to cell growth andviability that does not interfere with expression or stability of theproduced polypeptide or protein.

The temperature of the cell culture in the initial growth phase will beselected based primarily on the range of temperatures at which the cellculture remains viable. For example, during the initial growth phase,CHO cells grow well at 37° C. In general, most mammalian cells grow wellwithin a range of about 35° C. to 39° C. In one embodiment of theinvention, the temperature for growth phase (day 0 to day 3) is 37° C.,and the temperature for production phase (after day 3) is 31° C. Thoseof ordinary skill in the art will be able to select appropriatetemperature or temperatures in which to grow cells, depending on theneeds of the cells and the production requirements of the practitioner.

In one embodiment of the present invention, the temperature of theinitial growth phase is maintained at a single, constant temperature. Inanother embodiment, the temperature of the initial growth phase ismaintained within a range of temperatures. For example, the temperaturemay be steadily increased or decreased during the initial growth phase.Alternatively, the temperature may be increased or decreased by discreteamounts at various times during the initial growth phase. One ofordinary skill in the art will be able to determine whether a single ormultiple temperatures should be used, and whether the temperature shouldbe adjusted steadily or by discrete amounts.

The cells may be grown during the initial growth phase for a greater orlesser amount of time, depending on the needs of the practitioner andthe requirement of the cells themselves. In one embodiment, the cellsare grown for a period of time sufficient to achieve a viable celldensity that is a given percentage of the maximal viable cell densitythat the cells would eventually reach if allowed to grow undisturbed.For example, the cells may be grown for a period of time sufficient toachieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent or more ofmaximal viable cell density.

In another embodiment the cells are allowed to grow for a defined periodof time. For example, depending on the starting concentration of thecell culture, the temperature at which the cells are grown, and theintrinsic growth rate of the cells, the cells may be grown for 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moredays. In some cases, the cells may be allowed to grow for a month ormore. The cells would be grown for 0 days in the production bioreactorif their growth in a seed bioreactor, at the initial growth phasetemperature, was sufficient that the viable cell density in theproduction bioreactor at the time of its inoculation is already at thedesired percentage of the maximal viable cell density. The practitionerof the present invention will be able to choose the duration of theinitial growth phase depending on polypeptide or protein productionrequirements and the needs of the cells themselves.

The cell culture may be agitated or shaken during the initial culturephase in order to increase oxygenation and dispersion of nutrients tothe cells. In accordance with the present invention, one of ordinaryskill in the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor during theinitial growth phase, including but not limited to pH, temperature,oxygenation, etc. For example, pH can be controlled by supplying anappropriate amount of acid or base, and oxygenation can be controlledwith sparging devices that are well known in the art.

Shifting Culture Conditions

At the end of the initial growth phase, a culture condition(s) may beshifted so that a second set of culture conditions is applied and ametabolic shift occurs in the culture. The accumulation of inhibitorymetabolites, most notably lactate and ammonia, inhibits growth. Ametabolic shift, accomplished by, e.g., a change in the temperature, pH,osmolality or chemical inductant level of the cell culture, may becharacterized by, e.g., a reduction in the ratio of a specific lactateproduction rate to a specific glucose consumption rate. In onenonlimiting embodiment, the culture conditions are shifted by changingthe temperature of the culture. In another embodiment of the invention,the temperature shift occurs on day 1-7. In another embodiment of theinvention, the temperature is shifted to 29° C.-32° C. In anotherembodiment of the invention, the temperature shift occurs at day 3, andthe temperature is shifted to 31° C. Teachings regarding temperatureshift and metabolic shift may be found in the art (see, e.g., U.S.Published Patent Application No. US 2006/0121568).

Subsequent Production Phase

Once the conditions of the cell culture have been shifted as discussedabove, the cell culture may be maintained for a subsequent productionphase under a second set of culture conditions conducive to the survivaland viability of the cell culture and appropriate for expression of thedesired polypeptide or protein at adequate, e.g., commercially adequate,levels.

As discussed above, the culture may be shifted by shifting one or moreof a number of culture conditions including, but not limited to,temperature, pH, osmolality, and sodium butyrate levels. In oneembodiment, the temperature of the culture is shifted. According to thisembodiment, during the subsequent production phase, the culture ismaintained at a temperature or temperature range that is lower than thetemperature or temperature range of the initial growth phase. Forexample, during the subsequent production phase, CHO cells expressrecombinant polypeptides and proteins well within a range of 25° C. to35° C. In one embodiment of the invention, the production phase beginsafter day 3. In another embodiment of the invention, the productionphase is carried out at 31° C. As discussed in U.S. Published PatentApplication No. US 2006/0121568, multiple discrete temperature shiftsmay be employed to increase cell density or viability or to increaseexpression of the recombinant polypeptide or protein.

In accordance with the formula of the present invention, a desired cellmass (e.g., cell density) and production titer (e.g., target titer) areselected in order to establish the baseline-adjusted amino acidconcentration, A, and the starting amino acid concentration, B. Thus,generally the cells are maintained in the subsequent production phaseuntil the desired cell density or production titer, or a value(s) nearthe desired cell density or production titer is reached. In oneembodiment, the cells are maintained in the subsequent production phaseuntil the titer of the recombinant polypeptide or protein reaches amaximum. In other embodiments, the culture may be harvested prior tothis point, depending on the production requirement of the practitioneror the needs or viability of the cells themselves. For example, thecells may be maintained for a period of time sufficient to achieve aviable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95 or 99 percent or more of maximal viable celldensity. In some cases, it may be desirable to allow the viable celldensity to reach a maximum, and then allow the viable cell density todecline to some level before harvesting the culture. In an extremeexample, it may be desirable to allow the viable cell density toapproach or reach zero before harvesting the culture.

In another embodiment of the present invention, the cells are allowed togrow for a defined period of time during the subsequent productionphase. For example, depending on the concentration of the cell cultureat the start of the subsequent growth phase, the temperature at whichthe cells are grown, and the intrinsic growth rate of the cells, thecells may be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more days. In some cases, the cells may beallowed to grow for a month or more. The practitioner of the presentinvention will be able to choose the duration of the subsequentproduction phase depending on polypeptide or protein productionrequirements and the needs of the cells themselves. The duration ofculture will help determine the amino acid concentration required forcell maintenance, which may range in the present invention from, e.g.,0% to 150% of the amino acid concentration required for cell mass.

In certain cases, it may be beneficial or necessary to supplement thecell culture, i.e., feed the cell culture, during the subsequentproduction phase with nutrients or other medium components that havebeen depleted or metabolized by the cells. For example, it might beadvantageous to supplement the cell culture with nutrients or othermedium components observed to have been depleted during monitoring ofthe cell culture (see “Monitoring Cell Culture Conditions” section,below). Alternatively or additionally, it may be beneficial or necessaryto supplement the cell culture prior to the subsequent production phase.As nonlimiting examples, it may be beneficial or necessary to supplementthe cell culture with hormones and/or other growth factors, particularions (such as sodium, chloride, calcium, magnesium, and phosphate),buffers, vitamins, nucleosides or nucleotides, trace elements (inorganiccompounds usually present at very low final concentrations), lipids,amino acids, or glucose (or another energy source).

These supplementary components may all be added, i.e., fed, to the cellculture at one time, or they may be provided to the cell culture in aseries of additions. In one embodiment of the present invention, thesupplementary components are provided to the cell culture at multipletimes in proportional amounts. In another embodiment, it may bedesirable to provide only certain of the supplementary componentsinitially, and provide the remaining components at a later time. In yetanother embodiment of the present invention, the cell culture is fedcontinually with these supplementary components.

In accordance with the present invention, the total volume added to thecell culture should optimally be kept to a minimal amount. For example,the total volume of the feeding medium, or solution containing thesupplementary components, added to the cell culture may be 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume ofthe cell culture prior to providing the supplementary components. Thus,the feeding medium should be concentrated in order to avoid bioreactoroverflow or medium component dilution. The feeding medium is preferablyprovided to the main culture with the same pH, temperature, etc., butwith high concentrations of nutrients relative to the starting medium.In one embodiment of the invention, the feeding medium is the feedingmedium designated “Feed Medium” in column 4 of Table 2.

In one embodiment of the invention, a cell culture with a starting aminoacid concentration, B, is supplemented with an additional amino acid(s)in order to achieve a baseline-adjusted amino acid concentration, A. Inanother embodiment of the invention, the cell culture is provided with acontinuous feed from about days 3-21, or periodic feeds every 2-3 days.In another embodiment of the invention, the feeding occurs from aboutday 3 to about day 20 (for a 21-day culture) as bolus feeds. In anotherembodiment of the invention, the feeding occurs as periodic feeds aboutevery 2-3 days. In yet another embodiment of the invention, the feedingvolume is about 1% to about 40% of the total cell culture volume.

The cell culture may be agitated or shaken during the subsequentproduction phase in order to increase oxygenation and dispersion ofnutrients to the cells. In accordance with the present invention, one ofordinary skill in the art will understand that it can be beneficial tocontrol or regulate certain internal conditions of the bioreactor duringthe subsequent growth phase, including but not limited to pH,temperature, oxygenation, etc. For example, pH can be controlled bysupplying an appropriate amount of acid or base, and oxygenation can becontrolled with sparging devices that are well known in the art.

Monitoring Cell Culture Conditions

In certain embodiments of the present invention, the practitioner mayfind it beneficial or necessary to periodically monitor particularconditions of the growing cell culture. Monitoring cell cultureconditions allows the practitioner to determine whether the cell cultureis producing the recombinant polypeptide of interest at suboptimallevels or whether the culture is about to enter into a suboptimalproduction phase. In order to monitor certain cell culture conditions,it may be necessary to remove small aliquots of the culture foranalysis. One of ordinary skill in the art will understand that suchremoval may potentially introduce contamination into the cell culture,and will take appropriate care to minimize the risk of suchcontamination.

As nonlimiting examples, it may be beneficial or necessary to monitortemperature, pH, cell density, cell viability, integrated viable celldensity, lactate levels, ammonium levels, osmolarity, or titer of theexpressed polypeptide. Numerous techniques are well known in the artthat will allow one of ordinary skill in the art to measure theseconditions. For example, cell density may be measured using ahemacytometer, a Coulter counter, or cell density examination (CEDEX®,Innovatis, Malvern, Pa.). Viable cell density may be determined bystaining a culture sample with Trypan blue. Since only dead cells takeup the Trypan blue (i.e., viable cells exclude the dye), viable celldensity can be determined by counting the total number of cells,dividing the number of cells that take up the dye by the total number ofcells, and taking the reciprocal. HPLC can be used to determine thelevels of lactate, ammonium or the expressed polypeptide or protein.Alternatively, the level of the expressed polypeptide or protein can bedetermined by standard molecular biology techniques such as Coomassiestaining of SDS-PAGE gels, Western blotting, Bradford assays, Lowryassays, biuret assays, and UV absorbance. It may also be beneficial ornecessary to monitor the post-translational modifications of theexpressed polypeptide or protein, including phosphorylation andglycosylation.

Harvesting Polypeptides Produced by Cell Culture

The polypeptide of interest that is produced by the cell culture maythen be purified from the culture medium or from cell extracts for usein various applications. Soluble forms of the polypeptide can bepurified from conditioned media. Membrane-bound forms of the polypeptidecan be purified by preparing a total membrane fraction from theexpressing cell and extracting the membranes with a nonionic detergentsuch as TRITON® X-100 (EMD Biosciences, San Diego, Calif.). Cytosolic ornuclear proteins may be prepared by lysing the host cells (viamechanical force, Parr-bomb, sonication, detergent, etc.), removing thecell membrane fraction by centrifugation, and retaining the supernatant.

The polypeptide can be purified using other methods known to thoseskilled in the art. For example, a polypeptide produced by the disclosedmethods can be concentrated using a commercially available proteinconcentration filter, for example, an AMICON® or PELLICON®ultrafiltration unit (Millipore, Billerica, Mass.). Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin (e.g., a MonoQ column, Amersham Biosciences, Piscataway, N.J.) maybe employed; such resin contains a matrix or substrate having pendantdiethylaminoethyl (DEAE) or polyethylenimine (PEI) groups. The matricesused for purification can be acrylamide, agarose, dextran, cellulose orother types commonly employed in protein purification. Alternatively, acation exchange step may be used for purification of proteins. Suitablecation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups (e.g., S-SEPHAROSE® columns,Sigma-Aldrich, St. Louis, Mo.).

The purification of the polypeptide from culture supernatant may alsoinclude one or more column steps over affinity resins, such asconcanavalin A-agarose, AF-HEPARIN650, heparin-TOYOPEARL® or Cibacronblue 3GA SEPHAROSE® (Tosoh Biosciences, San Francisco, Calif.);hydrophobic interaction chromatography columns using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinity columnsusing antibodies to the labeled protein. Finally, one or more highperformance liquid chromatography (HPLC) steps employing hydrophobicHPLC media, e.g., silica gel having pendant methyl or other aliphaticgroups (e.g., Ni-NTA columns), can be employed to further purify theprotein. Alternatively, the polypeptides may be recombinantly expressedin a form that facilitates purification. For example, the polypeptidesmay be expressed as a fusion with proteins such as maltose-bindingprotein (MBP), glutathione-S-transferase (GST), or thioredoxin (TRX).Kits for expression and purification of fusion proteins are commerciallyavailable from New England BioLabs (Beverly, Mass.), Pharmacia(Piscataway, N.J.), and Invitrogen (Carlsbad, Calif.), respectively. Theproteins can also be tagged with a small epitope (e.g., His, myc or Flagtags) and subsequently identified or purified using a specific antibodyto the chosen epitope. Antibodies to common epitopes are available fromnumerous commercial sources.

As an alternative to traditional chromatography purification modes(e.g., flow-through and bind-elute chromatography purification modes),the polypeptides produced by the methods of the present invention may bepurified by operating a chromatography purification column in a weakpartitioning mode, a technique in which at least one product containedin the preparation, and at least one contaminant or impurity, both bindto a chromatographic resin or medium. In the weak partitioning mode, theat least one impurity binds more tightly to the medium than thepolypeptide product; and as loading (of the load fluid) continues,unbound polypeptide product selectively passes through the medium and isrecovered from the column effluent. Such purification results in a highdegree of impurity reduction, as well as high product recovery. Suchpurification can be achieved on media and resins known in the art,including but not limited to, charged ion exchange medium, hydrophobicinteraction chromatography resin, immobilized metal affinitychromatography resin, and hydroxyapatite resin. In at least oneembodiment, impurity/contaminant removal under weak partitioningconditions occurs as the load fluid passes through a medium/resin thatbinds at least 2.8 mg of product per ml of medium/resin. In at least oneother embodiment, impurity/contaminant removal under weak partitioningconditions occurs as the load fluid passes through a medium/resin atoperating conditions defined by a partition coefficient of at least 0.1.The purified product is recovered from the effluent of the columncontaining the medium/resin.

Table 5 summarizes the differences in the characteristics betweenflow-through (FT), bind-elute (B-E), and the weak partitioning (WP)modes.

TABLE 5 Characteristics of FT / WP / B-E Modes FT WP B-E Kp <0.10.1-20 >20 Load Impurities Impurities Product + impurities challenge10-50 mg Prod/mL 50-500 mg Prod/mL <100 mg Prod/mL limitation (typical)but actually (typical) but actually dependent on load purity dependenton load purity Load Vol. Moderate, for dilute Very high, for diluteLower, as the product binds impurities 10-20 CVs impurities up to 50 CVsin addition to impurities 5-20 CVs [Product] Equal to load Initial lag,then equal to <5% of load concentration in load concentration throughload concentration eluate much of load through much of load Residual LowVery low Dependent on elution [Impurity] conditions, pool volume andcapacity Product <1 mg/mL <10-20 mg/mL >10-20 mg/mL bound (Q) OperatingRelatively broad Modest window Stringent binding conditions region rangeof conditions of operation between for load, broad range of FT and B-Emodes elution conditions Mobile Isocratic Isocratic Change in bufferphase(s) composition after load which causes elution

The partition coefficient (Kp) is the ratio of the concentration of theadsorbed product (Q) to the concentration of the product in solution(C); thus the Kp for the weak partitioning mode is intermediate betweenthe Kp for the flow-through and bind-elute modes, e.g., between about0.1 and 20. In order to determine the proper conditions, e.g., salt,buffer, pH, etc., for a weak partitioning mode of purification, ahigh-throughput screen or a batch purification screening study can beperformed. Thus, one skilled in the art can determine product partitioncoefficients K_(p) as a function of operating conditions (see Example7.1).

Purification methods using a weak partitioning mode are described indetail in U.S. patent application Ser. Nos. 11/372,054 and 11/510,634,both of which are incorporated by reference herein in their entireties.

Some or all of the foregoing purification steps in various combinationsor with other known methods, can be employed to purify a polypeptide ofinterest produced by the large-scale animal cell culture methods andmedia described herein.

Pharmaceutical Compositions Containing Polypeptides Produced by CellCulture

The foregoing cell culture media, e.g., large-scale cell culture media,and methods of culturing cells provides polypeptides of interest, e.g.,antibodies, soluble receptors, fusion proteins, etc. The polypeptidesproduced by the disclosed cell culturing methods, and with the novelmedia and related methods disclosed herein, including antibodies andfragments thereof, may be used in vitro, ex vivo, or incorporated intopharmaceutical compositions and administered to individuals (e.g., humansubjects) in need thereof. Several pharmacogenomic approaches toconsider in determining whether to administer a polypeptide of theinvention are well known to one of skill in the art and includegenome-wide association, candidate gene approach, and gene expressionprofiling. A pharmaceutical composition of the invention is formulatedto be compatible with its intended route of administration (e.g., oralcompositions generally include an inert diluent or an edible carrier).Other nonlimiting examples of routes of administration includeparenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g.,inhalation), transdermal (topical), transmucosal, and rectaladministration. The pharmaceutical compositions compatible with eachintended route are well known in the art.

A polypeptide of the invention may be used as a pharmaceuticalcomposition when combined with a pharmaceutically acceptable carrier.Such a composition may contain, in addition to a polypeptide of theinvention, carriers, various diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials well known in the art.The term “pharmaceutically acceptable” means a nontoxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredient(s). The characteristics of the carrier will dependon the route of administration.

The pharmaceutical composition of the invention may also containadditional therapeutic factors or agents for treatment of the particulartargeted disorder. For example, a pharmaceutical composition fortreatment of type 2 diabetes may also include an oral antidiabeticagent. The pharmaceutical composition may contain thrombolytic orantithrombotic factors such as plasminogen activator and Factor VIII.The pharmaceutical composition may further contain anti-inflammatoryagents. Such additional factors and/or agents may be included in thepharmaceutical composition to produce a synergistic effect with apolypeptide of the invention, or to minimize side effects caused by thepolypeptides of the invention.

The pharmaceutical composition of the invention may be in the form of aliposome in which a polypeptide of the invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids that exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, etc.

As used herein, the term “therapeutically effective amount” means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, e.g.,amelioration of symptoms of, healing of, or increase in rate of healingof such conditions. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of a polypeptide of the invention isadministered to a subject, e.g., a mammal (e.g., a human). A polypeptideof the invention may be administered in accordance with the method ofthe invention either alone or in combination with other therapies. Whencoadministered with one or more agents, a polypeptide of the inventionmay be administered either simultaneously with the second agent, orsequentially. If administered sequentially, the attending physician willdecide on the appropriate sequence of administering the polypeptides ofthe invention in combination with other agents.

When a therapeutically effective amount of a polypeptide of theinvention is administered orally, the binding agent will be in the formof a tablet, capsule, powder, solution or elixir. When administered intablet form, the pharmaceutical composition of the invention mayadditionally contain a solid carrier such as a gelatin or an adjuvant.The tablet, capsule, and powder contain from about 5 to 95% bindingagent, and preferably from about 25 to 90% binding agent. Whenadministered in liquid form, a liquid carrier such as water, petroleum,oils of animal or plant origin, such as peanut oil (exercising cautionin relation to peanut allergies), mineral oil, soybean oil, or sesameoil, or synthetic oils may be added. The liquid form of thepharmaceutical composition may further contain physiological salinesolution, dextrose or other saccharide solution, or glycols such asethylene glycol, propylene glycol, or polyethylene glycol. Whenadministered in liquid form, the pharmaceutical composition containsfrom about 0.5% to about 90% by weight of the binding agent, andpreferably from about 1% to about 50% by weight of the binding agent.

When a therapeutically effective amount of a polypeptide of theinvention is administered by intravenous, cutaneous or subcutaneousinjection, the polypeptide of the invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill of thosein the art. A preferred pharmaceutical composition for intravenous,cutaneous, or subcutaneous injection should contain, in addition to thepolypeptide of the invention, an isotonic vehicle such as sodiumchloride injection, Ringer's injection, dextrose injection, dextrose andsodium chloride injection, lactated Ringer's injection, or other vehicleas known in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or any other additive(s) known to those of skill in theart.

The amount of a polypeptide of the invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments that the patient has undergone. Ultimately, the attendingphysician will decide the amount of a pharmaceutical composition orpolypeptide of the invention with which to treat each individualpatient. Initially, the attending physician will administer low doses ofa pharmaceutical composition or polypeptide of the invention and observethe patient's response. Larger doses of a pharmaceutical composition orpolypeptide of the invention may be administered until the optimaltherapeutic effect is obtained for the patient, and at that point thedosage is not generally increased further. It is contemplated that thevarious pharmaceutical compositions used to treat a subject in needthereof should contain about 0.1 μg to about 100 mg of a polypeptide ofthe invention per kg body weight.

The duration of intravenous (i.v.) therapy using a pharmaceuticalcomposition of the present invention will vary, depending on theseverity of the disease being treated and the condition and potentialidiosyncratic response of each individual patient. It is contemplatedthat the duration of each application of a pharmaceutical composition ora polypeptide of the present invention may be within the range of, e.g.,1-12, 6-18, or 12-24 hrs of continuous or intermittent i.v.administration. Also contemplated is subcutaneous (s.c.) therapy using apharmaceutical composition of the present invention. These therapies canbe administered daily, weekly, or, more preferably, biweekly, ormonthly. Ultimately the attending physician will decide on theappropriate duration of i.v. or s.c. therapy, or therapy with a smallmolecule, and the timing of administration of the therapy, using thepharmaceutical composition of the present invention.

All of the references, patents, patent applications, and publicationscited in this application are hereby incorporated by reference herein intheir entireties.

EXAMPLES

The Examples which follow are set forth to aid in the understanding ofthe invention but are not intended to, and should not be construed to,limit the scope of the invention in any way. The Examples do not includedetailed descriptions of conventional methods, such as recombinant DNAtechniques. Such methods are well known to those of ordinary skill inthe art.

Example 1 Quantifying the Amino Acid Composition of CHO Cells

To quantify the amino acid composition of antibody-expressing CHO cellsand recombinant protein-expressing CHO cells, i.e., the molar percentageof each amino acid relative to the amino acid total of cell mass(biomass), the following procedure was performed. Briefly, three CHOcell lines overexpressing antibodies or recombinant protein, morespecifically anti-IL-22 antibody (recombinant human anti-IL-22antibody), Myo-029 antibody (anti-GDF8 IgG1 monoclonal antibody), andrecombinant human BMP-2, were grown in a shake flask for one (BMP-2) orthree (Myo-029 and anti-IL-22) days. On the final day, the cultures wereharvested and the cells spun to a concentration of 10⁶ cells/mL. A 10⁶cell-containing pellet was washed twice with 1×PBS and the pelletresuspended in 500 μL of 5 N HCl. The cell-containing suspension washeated at 100° C. for 24 hours, at which point the suspension was vacuumcentrifuged. The resulting pellet was resuspended in 500 μL PBS, andamino acid analysis was performed using gas or liquid chromatography.

Acid hydrolysis determined that methionine and tryptophan both degradeduring acid hydrolysis; thus, the concentrations of these amino acids inExamples 2 and 3 were based on literature values for CHO cells. Inaddition, it was determined that glutamine and asparagine were convertedto their acidic forms, glutamic acid and aspartic acid, respectively,during hydrolysis; thus, the concentrations for these four amino acidsin Examples 2 and 3 were adjusted based on the glutamine/glutamic acidand asparagine/aspartic acid ratios reported in literature. Otherwise,it was determined that these CHO cell lines possess similar amino acidcompositions, and closely matched reported values for other mammaliancells (data not shown) (see Bonarius (1996) Biotechnol. Bioeng.50:299-318).

Example 2 Desired Peak Cell Density of 15×10⁶ cells/ml, and TargetAnti-IL-22 Antibody Titer of 9 g/L in CHO Cells (Cell Line 1) with 32%Volume Feed Using Rationally Designed Medium Example 2.1 RationallyDesigned Medium

Using the formula disclosed herein, the baseline-adjusted amino acidconcentration, A, of amino acids required for 15×10⁶ cells/ml producing9 g/L anti-IL-22 antibody in CHO Cell Line 1 were determined (Table 6,column 2) (cell maintenance was set at 50%, i.e., Y=0.5). Thebaseline-adjusted amino acid concentration, A (Table 6, column 2), of avariety of amino acids were adjusted to obtain the modifiedbaseline-adjusted total amino acid concentrations shown in column 3 ofTable 6. The following adjustments were made: levels of Asn, Asp, andGln were set according to U.S. Published Patent Application No.US2006/0121568A1; 2) because Ala, Glu, Gly were produced by the cellcultures, their levels were adjusted to lower values; 3) the level ofCys was adjusted due to the fact that cystine is also used in thefeeding medium, and the medium value of cystine is set to that in U.S.Published Patent Application No. US2006/0121568A1; 4) levels of Arg,His, Ile, Leu, Lys, Met, Phe are 20% to 100% higher than thebaseline-adjusted amino acid concentration, A, due to the fact that thefeed powder, which contains a set amino acid composition, was used tomake the desired cell culture medium, such that an exact match was notpossible. The starting medium amino acid concentration, B (Table 6,column 4), was calculated from the modified baseline-adjusted totalamino acid concentrations shown in column 3 of Table 6.

TABLE 6 Desired Cell Culture Medium Formulation Baseline-AdjustedModified Baseline- Starting Amino Acid Adjusted Amino Acid ConcentrationAmino Acid Concentration Amino Acid (mM) (A) Concentration (mM) (mM) (B)ALA 13.67 2.18 0.20 ARG 6.68 12.56 1.94 ASN 7.42 27.82 14.56 ASP 11.866.28 1.70 CYS 4.97 0.27 0.40 GLU 9.23 2.18 0.20 GLN 13.11 2.72 4.00 GLY12.94 4.52 3.63 HIS 3.93 5.05 2.15 ILE 6.23 10.95 2.54 LEU 13.13 17.966.87 LYS 13.01 15.62 7.91 MET 2.82 5.71 2.38 PHE 6.19 7.67 3.76 PRO11.50 11.15 7.36 SER 21.35 22.34 10.20 THR 13.89 14.18 8.80 TRP 3.002.89 1.84 TYR 7.53 7.55 5.10 VAL 15.44 15.24 10.36 Total 197.91 194.8595.91

Example 2.2 Cell Density and Antibody Titer in Response to RationallyDesigned Medium

Cell line 1 cells (anti-IL-22-expressing CHO cells) were obtained fromshake flasks containing day 3 cultures, and were inoculated at 0.7×10⁶cells/ml on day 0 into the starting cell culture medium in a 1 Lbioreactor (Applikon 2 L, (Applikon Inc, Foster City, Calif.)). On day 3(about 80 hours), temperature was shifted from 37° C. to 31° C. and FeedMedium (see column 4 of Table 2) was added at 3.75%, 4%, 4%, 9%, 2%, 1%,1%, 1%, 3%, 2% and 1% by volume on days 3, 5, 6, 7, 10, 11, 12, 13, 14,17, 20, respectively, to obtain the desired cell culture medium, whichcontains the amino acid concentration shown in Table 6, column 3. Cellcultures were maintained at pH 7.0, in a dissolved oxygen level of 30%,and with agitation at 200 rpm. Samples were taken daily to test for celldensity (CEDEX® cell counting instrument (Innovatis, Malvern, Pa.)),viability (CEDEX®) and certain metabolite levels (Nova BioProfileAnalyzer, Nova Biomedical Cooperation, Waltham, Mass.). Spun-down mediawere saved at −80° C. for antibody titer analysis using Protein A HPLC.

The results are shown in FIGS. 1 and 2. As can be seen from FIG. 1, thehighest cell density (about 11×10⁶ cells/mL) was achieved on day 11 ofculture, with cell density decreasing thereafter. The highest antibodytiter (about 7 g/L) was obtained on day 21 of culture. Thus, therationally designed medium may be used to produce high cell density andhigh antibody titer.

Example 3 Desired Peak Density of 15×10⁶ cells/ml, and Target Anti-IL-22Antibody Titer of 10 g/L in CHO Cells (Cell Line 2) with 33% Volume FeedUsing Rationally Designed Medium Example 3.1 Rationally Designed Medium

Using the formula disclosed herein, the baseline-adjusted amino acidconcentration, A, of amino acids required for 15×10⁶ cells/ml and 10 g/Lanti-IL-22 antibody (described above) were determined (cell maintenancewas set at 50%, i.e., Y=0.5). The baseline-adjusted amino acidconcentration, A (Table 7, column 2) of a variety of amino acids wereadjusted as described in Example 2 to obtain the modifiedbaseline-adjusted total amino acid concentrations shown in column 3 ofTable 7. In addition, for this medium formulation both the starting cellculture medium and the feed medium were prepared from existing powderswith fixed compositions, and thus an exact match was not possible. Thestarting medium amino acid concentration, B (Table 7, column 4), wascalculated from the modified baseline-adjusted total amino acidconcentrations shown in column 3 of Table 7.

TABLE 7 Desired Cell Culture Medium Formulation Baseline-AdjustedModified Baseline- Starting Amino Acid Adjusted Amino Amino Acid AminoConcentration Acid Concentration Acid (mM) (A) Concentration (mM) (mM)(B) ALA 14.2 2.4 0.4 ARG 6.9 15.2 5.3 ASN 7.8 32.6 21.1 ASP 12.2 6.8 2.3CYS 5.2 0.3 0.4 GLU 9.7 2.4 0.4 GLN 13.6 2.7 4.0 GLY 13.6 4.5 3.6 HIS4.1 5.5 2.7 ILE 6.4 13.2 5.4 LEU 13.8 20.0 9.4 LYS 13.7 16.5 8.9 MET 2.96.3 3.1 PHE 6.5 8.3 4.5 PRO 12.3 12.5 9.1 SER 22.6 23.8 11.8 THR 14.815.7 10.8 TRP 3.2 3.2 2.3 TYR 8.0 7.6 5.1 VAL 16.4 15.4 10.3 Total207.93 214.8 121.1

Example 3.2 Cell Density and Antibody Titer in Response to RationallyDesigned Medium

Cell line 2 cells (anti-IL-22-expressing CHO cells) were obtained fromshake flasks containing day 3 cultures, and the cells were inoculated at0.7×10⁶ cells/ml on day 0 into the starting cell culture medium in a 1 Lbioreactor (Applikon 2 L). On day 3 (about 80 hours), temperature wasshifted from 37° C. to 31° C. and feed was added continuously (1.8% byvolume per day) with an automatic feeding pump, to obtain the desiredcell culture medium, which contains the amino acid concentration shownin Table 7, column 3. Cell cultures were maintained at pH 7.0, in adissolved oxygen level of 30%, and with agitation at 200 rpm. Sampleswere taken daily to test for cell density (CEDEX® cell countinginstrument), viability (CEDEX®) and certain metabolites levels (NovaEnzymatic analyzer). Spun-down media were saved at −80° C. for antibodytiter analysis using Protein A HPLC.

The results are shown in FIGS. 3 and 4. As can be seen from FIG. 3, thehighest cell density (over 12×10⁶ cells/mL) was achieved on day 10 ofculture, with cell density decreasing thereafter. The highest antibodytiter (over 10 g/L) was obtained on day 19 of culture. Thus, therationally designed medium may be used to produce high cell density andhigh antibody titer.

Example 4 The Effect of Proline Addition on Cell Culture Performance

CHO cells producing the antibody Anti-IL-22 were cultured inpH-controlled shake flasks (500 mL flasks with 100 mL working volume),and maintained on a shaker at ˜100 rpm in a temperature-controlled, 7%CO₂ incubator. Cells were seeded at 0.7×10⁶ cells/mL. The cultureduration was 18 days. The first 3 days cells were maintained at 37° C.,after which the temperature was shifted to 31° C. for the remainder ofthe culture. pH was controlled for the first 3 days with 1 M sodiumbicarbonate. Cell culture medium consisted of a medium based ontraditional cell culture requirements, termed “Traditional Medium,” andrationally designed medium, i.e., medium formulated using the rationaldesign disclosed herein, termed “Rational Design Medium,” to achieve10×10⁶ cells/mL and 10 g/L antibody. A major difference of note betweenthese two formulations is that several amino acids (PRO, THR, GLY, TYR,TRP, VAL, and PHE) are at higher concentrations in the “Rational DesignMedium” compared to the “Traditional Medium.” A third conditionpresented is the traditional medium with an additional 3.7 mM prolineadded to obtain the level of proline in the “Rational Design Medium”,termed “Traditional Medium+Proline.” These formulations are presented inTable 8, below. Wyeth in-house feed medium (“Feed Medium,” see Table 2)was added to the culture at 23% total by volume, comprising daily feedsof 2% on days 3-4, 9-14, 17, 1% on days 5-6, and 3% on day 7.

TABLE 8 Media Formulations for Proline Studies 1 2 3 4 RationalTraditional Traditional Design Medium Medium Medium + Proline Amino Acid[mM] [mM] [mM] alanine 0.44 0.44 0.44 arginine•HCl 5.32 5.32 5.32asparagine•H₂O 21.08 21.08 21.08 aspartic acid 2.25 2.25 2.25 glutamine4.00 4.00 4.00 glutamate 0.24 0.24 0.24 glycine 1.78 3.59 1.78 histidine2.68 2.68 2.68 isoleucine 5.44 5.44 5.44 leucine 9.43 9.43 9.43lysine•HCl 8.90 8.90 8.90 methionine 3.08 3.08 3.08 phenylalanine 3.674.48 3.67 proline 5.41 9.13 9.13 serine 11.82 11.82 11.82 threonine 5.7110.85 5.71 tryptophan 1.54 2.32 1.54 tyrosine•2Na 3.34 5.13 3.34 valine7.36 10.30 7.36

The results from these experiments are shown in FIGS. 5-7. Addition ofproline to the traditional medium, i.e. “Traditional Medium+Proline”(FIG. 5), resulted in an antibody production equivalent to the “RationalDesign Medium” through day 14. As shown in FIG. 6, all three mediamaintained a high cell density, with the highest density displayed atday 12. As shown in FIG. 7, all three media maintained high cellviability, with the “Rational Design Medium” cultures maintaininggreater viability on days 15-18 in comparison to the cultures containing“Traditional Medium” and “Traditional Medium+Proline” media. As avariety of other media, which each contained one rationally designedconcentration of either glycine, phenylalanine, threonine, tryptophan,tyrosine or valine did not result in cell cultures producing a higherantibody titer than the “Traditional Medium” (data not shown), prolineserves as the rate-limiting amino acid required to achieve high titer.This is exemplified in FIGS. 8A-E, which show that by day 14 many of theamino acids in the “Traditional Medium+Praline” reached extremely lowlevels (note tyrosine was depleted), thereby preventing furtherincorporation of these amino acids into antibody. This result is alsoobserved in the titer graph (FIG. 5), as the slope for the “TraditionalMedium+Proline” is reduced after day 14, while antibody production ismaintained through day 18 for the “Rational Design Medium,” whichcontains higher levels of other amino acids.

Interestingly, the proline concentration in the “Traditional Medium”never dropped below 1 mM (FIG. 8A); however, the effect of proline onoverall amino acid incorporation into antibody was reduced after day 11.This finding suggests that a proline threshold exists, i.e., theconcentration of proline must be maintained above 1 mM throughout theculture.

Example 5 Prophetic Example Optimized Cell Culture Media for a NovelCell Line

The methods of media design disclosed herein may be used for any cellculture, including cell cultures that use novel cells/cell lines. Theoptimized media for use with a novel cell line would contain at leastone baseline-adjusted amino acid concentration, A, of an amino acidaccording to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F.

A multiplier, M, when applying the above equation to a novel cellculture, may be selected, e.g., from 1 to 20×10⁶ cells/mL. One of skillin the art would be able to calculate a useful M value by doubling themaximum cell density during the growth phase, which can be calculatedbased on the growth rate.

A multiplier, N, when applying the above equation to a novel cellculture, may be calculated, e.g., by multiplying the IVC by the cell qp.One of skill in the art would be able to calculate the IVC by estimatingthe growth profile, following the methods described in the sectionentitled “Rational Media Design and Formulations.” One of skill in theart would be able to calculate the qp by measuring the antibody orrecombinant protein production on a per cell basis.

A cell maintenance factor, Y, when applying the above equation to anovel cell culture, may be estimated by using Y=1 (i.e., 100% of theamino acid(s) required for desired cell mass) initially, and thenrefining the value of Y (higher or lower).

A baseline factor, F, when applying the above equation to a novel cellculture, may be estimated by using F=1.3 (i.e., 30% additional aminoacid(s)) initially, and then refining the value of F (higher or lower).

Example 6 Effect of Maintenance and Baseline Factors on Cell CulturePerformance

To demonstrate that the maintenance factor, Y, and the baseline factor,F, are important/essential for cell culture performance,anti-IL-22-expressing CHO cells were seeded at 0.7×10⁶ cells/mL andcultured for 21 days in 2 L bioreactors with a pH set point of 7.0 and adissolved oxygen (DO) setpoint of 30%. The pH was controlled by 2NNa₂CO₃/NaHCO₃, and DO was controlled by air (containing 7% CO₂) sparge.The temperature of the culture was 37° C. for the first 3 days andshifted to 31° C. after day 3, and remained at 31° C. until the end ofthe culture. Cells were cultured for 21 days in either (1) mediacontaining amino acids required for both target protein titer anddesired peak cell density (but not accounting for the maintenance factoror baseline factor), i.e., “Rational Design Medium without maintenanceand baseline factors,” or (2) “Rational Design Medium.”

As exemplified in FIGS. 9 and 10, the medium that does not account formaintenance and baseline factors displayed a significant decrease inviability, nearly approaching 0% at day 21 of the cell culture, and asignificant decrease in cell density. Moreover, FIG. 11 demonstratesthat at day 21 of the cell culture, this same medium was only able tosupport an antibody titer of 5 g/L.

In contrast, Rational Design Medium (including maintenance and baselinefactors) displayed higher viability, cell density and antibody titers(FIGS. 9-11). At day 21 of the cell culture, the Rational Design Mediumwas able to support an antibody titer of 10 g/L.

These findings suggest that accounting for maintenance and baselinefactors in determination of the amino acid concentration in the desiredcell culture medium improves cell performance as measured by cellviability, cell density, and polypeptide titer.

Example 7 Polypeptide Purification Using Anion Exchange Chromatographyin a Weak Partitioning Mode Example 7.1 High-Throughput Screen toEstablish Weak Partitioning and Flow-Through Conditions

An initial screening study was performed first, which determined thepartition coefficient and/or the concentration of product bound to theresin under various solution conditions, thus defining the operatingregions of weak partitioning (WP) and flow-through (FT) modes forMab-AAB, the polypeptide of interest, and TMAE-HiCap (M) Medium. Thisscreen varied the concentration of sodium chloride and pH to determinetheir effects on the extent of binding of Mab-AAB and process-relatedimpurities (Protein A and HCP) to the TMAE medium.

The levels of Protein A residuals in the test samples were measuredusing a Protein A enzyme-linked immunosorbent assay (ELISA). The amountof high molecular weight aggregate was measured using an analytical sizeexclusion chromatography (SEC) assay. The levels of host cell proteins(HCPs) were measured using a HCP ELISA. All screening and column studieswere conducted at room temperature.

Fifty μL of TMAE HiCap medium was added to each well of a 96-well filterplate. Each well was equilibrated in solutions made up 50 mM glycine anda variable amount of Tris buffer (depending upon the amount needed forneutralization to the pH specified in Table 9) and sodium chloride(specified in Table 10). The pH ranged from 7.6 to 9.0, and the sodiumchloride ranged from 0 mM to 80 mM.

The buffer solutions used in each row were diluted on an automatedpipetting system (Tecan 100 RST). The stock solution for the buffers wasmade from 500 mM glycine acidified with HCl to pH 3.0, and subsequentlyneutralized with 2 M Tris Base to the pH levels indicated in Table 9.This titration resulted in a level of Tris that depended upon the pH ofthe buffer. The buffer pH was measured at a 1 to 10 dilution of thestock buffer concentration, which corresponded to the dilution made bythe automated pipetting system. As a result of the glycine acidificationto pH 3.0, the buffer contributes about 10 mM of ionic strength to thefinal solution. Two load (load fluid) challenges were made to the resin:5 mg/mL to measure the partition coefficient, Kp, and 122 mg/mL tomeasure the capacity of the resin for removal of impurities and thebound product, Q, in equilibrium with a protein solution at aconcentration approximately equal to the column load concentration.

TABLE 9 Buffer type and pH Target in Each Well All columns A 50 mMGlycine, 8.8 mM Tris, pH 7.6  B 50 mM Glycine, 13.6 mM Tris, pH 7.8 C 50mM Glycine, 16.0 mM Tris, pH 8.0 D 50 mM Glycine, 19.6 mM Tris, pH 8.2 E50 mM Glycine, 28.4 mM Tris, pH 8.4 F 50 mM Glycine, 37.2 mM Tris, pH8.6 G 50 mM Glycine, 64.0 mM Tris, pH 8.8 H 50 mM Glycine, 100 mM Tris,pH 9.0 

TABLE 10 NaCl levels (in mM) and Protein Challenges (mg/mL) in Each WellAll Rows 1 2 3 4 5 6 7 8 9 10 11 12 NaCl (mM) 0 10 20 40 60 80 0 10 2040 60 80 MAb-AAB 5 5 5 5 5 5 132 132 132 132 132 132 (mg/mL)

In the first stage of the high-throughput screen, each well wasequilibrated in the conditions of NaCl and pH as described in Tables 9and 10 in a phase volume ratio of 6:1 (300 uL solution:50 uL resin). Theplate was shaken for 20 minutes, allowing equilibrium to be reached. Thesolution was then removed by centrifuging the filter plate. Thisequilibration cycle was repeated three times.

In the second stage, the resin in each well was challenged with aconcentrated MAb-AAB solution to 5 mg/mL of resin with a volume ratio of6:1 (300 uL solution:50 uL resin) at the appropriate NaCl concentrationand pH. A 36 mg/mL solution of Mab-AAB in 1 mM HEPES, 10 mM NaCl, pH 7.0spiked with 300 ppm of Protein A was used as stock solution. The loadedplate was shaken for 20 minutes, allowing the resin and solution toequilibrate. The supernatant was removed from the filter plate bycentrifugation and collected into a collection plate. The proteinconcentration in the supernatant in each well was determined byabsorbance at A280 nm.

In the third stage, resin was washed by adding solutions of thespecified NaCl and pH conditions listed in Table 10. The supernatant wasremoved after shaking for 20 minutes. In the fourth stage, 2 M NaCl wasadded to remove the remaining protein that was bound to the resin. Thepartition coefficients were calculated for each well using the masseluted from stages 3 and 4 and the product concentration from stage 2,and are shown in Table 11.

TABLE 11 Partition Coefficients (Kp) for the 96-well HTS Screen forMAb-AAB 1 2 3 4 5 6 7 8 9 10 11 12 A 0.22 0.32 0.35 0.17 0.24 0.23 0.210.24 0.21 0.19 0.17 0.16 B 0.37 0.36 0.38 0.25 0.24 0.08 0.28 0.26 0.220.24 0.18 0.16 C 0.63 0.48 0.47 0.27 0.15 0.20 0.31 0.28 0.26 0.20 0.230.16 D 1.24 1.12 0.68 0.36 0.30 0.17 0.42 0.39 0.34 0.23 0.23 0.18 E3.24 1.89 1.05 0.59 0.35 0.15 0.68 0.58 0.41 0.29 0.21 0.18 F 8.37 3.371.56 0.61 0.31 0.32 0.87 0.74 0.51 0.32 0.25 0.21 G 18.36 9.49 3.16 0.820.49 0.34 0.91 0.88 0.69 0.39 0.24 0.20 H 125.73 23.79 6.58 1.23 0.580.43 1.18 1.02 0.78 0.42 0.27 0.24

As shown in Table 11, the Kp value can be used to describe regions whereMAb-AAB binds to the TMAE medium with different strengths. The strengthof MAb-AAB binding to the TMAE medium can be manipulated by varyingconditions of pH and chloride concentration into flow-through (K≦0.1),weak partitioning (0.1<K<20), and binding zones (K≧20).

The supernatant from the load stage of all wells from each zone weresampled and submitted for Protein A analysis. The assay results of thesesamples are summarized in Table 12. There is a region of pH andconductivity in which the TMAE chromatography step provides verysignificant removal of Protein A with limited protein loss to the resin.This region was found to be closely correlated to the partitioncoefficient value, Kp, and not any specific pH or chlorideconcentration.

TABLE 12 Protein A Log Removal Values (LRV) for MAb-AAB binding datafrom HTS Screen 1 2 3 4 5 6 7 8 9 10 11 12 A 2.11 1.89 2.12 1.85 1.221.00 1.63 1.02 1.00 0.92 0.85 1.02 B 2.79 2.37 2.42 1.96 1.23 1.13 1.771.81 1.22 0.85 0.94 1.52 C >3.05 >3.03 2.74 2.16 1.37 1.11 2.25 2.151.96 1.16 1.06 0.95 D >3.41 >2.98 >3.06 2.50 1.94 1.18 3.39 3.11 2.571.41 1.02 0.89 E >2.87 >2.93 >3.01 >2.95 2.13 1.75 >3.09 3.27 3.09 1.661.89 0.99 F >2.64 >2.89 >2.99 >3.11 2.29 1.82 >3.07 >3.11 >3.15 2.191.24 0.84 G >2.33 >2.58 >2.89 >3.07 2.41 2.14 >3.09 >3.11 >3.14 2.801.46 0.85 H >1.63 >2.36 >2.76 >3.01 2.86 2.37 >2.98 >3.05 >3.15 3.163.45 0.85

Example 7.2 Column Runs Under Flow-Through Conditions

The following experiment was performed in the flow-through (FT) mode,where the MAb-AAB interacts only very weakly with the column. Two runswere performed with load challenges of 110 mg/ml and 200 mg/ml of resin.

For all TMAE (HiCapM) anion exchange chromatography runs described, thefollowing conditions were used (exceptions are noted in the individualexperimental descriptions).

-   -   Operational flow rate—150-300 cm/hr    -   Equilibration 1—50 mM Tris, 2.0 M NaCl, pH 7.5 (5 column        volumes)    -   Equilibration 2—as specified, approximately equivalent to the        load pH and chloride content    -   Post-load wash—as specified, approximately equivalent to the        load pH and chloride content    -   Strip buffer—50 mM Tris, 2.0 M NaCl, pH 7.5 (5 column volumes)

Mabselect Protein A Chromatography

The culture containing the monoclonal antibody was purified at Pilotscale using a MabSelect column (2,389 mL) connected to a MilliporeK-prime 400 chromatography system. A Mabselect Protein A column wasequilibrated with 5 column volumes of 50 mM Tris/150 mM NaCl, pH 7.5 ata flow rate of 300 cm/hr. The column was then loaded at a load ofapproximately 40 mg product/ml resin. This was followed by a 5 columnvolume (CV) wash in 1 M NaCl, 50 mM Tris, pH 7.5, and a 5CV washcontaining 10 mM Tris, 75 mM NaCl, pH 7.5 wash. The column was theneluted using 50 mM glycine, 75 mM NaCl, pH 3.0. The product pool wasneutralized to pH 7.6 using 2 M Tris, pH 8.5. The neutralized peak had achloride concentration of approximately 90 mM.

TMAE HiCap (M) Chromatography

The neutralized Protein A pool was further purified over the TMAE stepwith the equilibration, load, and wash solutions at pH 7.5 with 50 mMTris and 75 mM sodium chloride. Five column volumes of wash were used.The column dimensions and load challenges for these two studies were:Run 1: 7.0 cm diameter×20.6 cm bed height (volume—793 mL) with a loadconcentration of 11.9 mg/mL; and Run 2: 7.0 cm diameter×13 cm bed height(volume—500 mL) with a load concentration of 17.6 mg/mL.

These load conditions were in the flow-through (FT) region (Table 13).Batch binding studies were used to measure the partition coefficient(Kp), and the bound product was determined by protein in the columnstrip by using UV absorbance. This method of determining the boundproduct typically underestimates the amount of product bound during theload due to isocratic elution of the product during the wash. The levelsof Protein A, HCP and high molecular weight aggregates (HMW) in the loadand product pool were measured, and the extent of removal calculated.The results are presented in Table 13. There is poor removal of ProteinA and HMW, and modest reduction in HCP levels.

TABLE 13 Removal of HCP, Protein A, and HMW under FT Conditions Par-tition Bound Re- Load Coef- Product Protein cov- Challenge ficient(mg/mL HCP A HMW ery Run (mg/mL) (Kp) resin) (LRV) (LRV) (Fold) (%) 1110 0.17 1.4 2.3 0.1 — 96 2 200 0.17 3.3 2.0 <0.1 1.5 96 *Impuritylevels were 38.5 ppm ProA and 51,943 ppm HCP (Run 1), 8.8 ppm ProA and25,398 ppm HCP (Run 2).

Example 7.3 Column Runs Under Weak Partitioning Conditions (High ProductChallenge) TMAE (HiCap M) Anion Exchange Chromatography

Several Mabselect Protein A runs were performed essentially as describedin Example 7.2 to generate the load material for these runs. Thepartially purified antibody pool from the Protein A step was furtherpurified over the TMAE column. The load to the TMAE column was in 50 mMTris, pH 8.2. The column diameter was 0.5 cm and the bed height was 10cm bed height (volume—2.0 mL). The column was challenged to a load of500 mg/mL resin, with a load concentration of 27.7 mg/mL.

The column was equilibrated with 5CV of a solution containing 50 mMTris, 2M NaCl, pH 7.5 followed by another equilibration step comprisinga 50 mM Tris, pH 8.2 solution. The column was then loaded to 500 mgproduct/ml resin with the neutralized Protein A peak from the previousstep and the product was recovered in the column effluent during theload cycle and some column volumes of the wash fraction.

These load conditions are in the weak partitioning region. Batch bindingstudies were used to measure the partition coefficient (Kp), and productbinding at high protein concentrations. At pH 8.2, and an approximatechloride content of 12 mM, the partition coefficient, Kp, is estimatedto be 1.9 (from interpolation of the dataset from the HTS screen).

The levels of HCP and Protein A were measured in three fractions duringthe loading stage representing load challenges of approximately 250,375, and 500 mg/ml of resin. The results from example 7.3 are presentedin Table 14. These results demonstrate that very high product challengescan be achieved in weak partitioning mode, without breakthrough ofimpurities. Excellent reduction in both HCP and Protein A was achieved,along with a 50% reduction in HMW content. In comparison to the resultsfor operation in the flow-through mode in Table 13, the removal ofimpurities was much better in the weak partitioning mode.

TABLE 14 Removal of HCP, Protein A and HMW for a 500 mg/ml TMAE loadchallenge Early Middle Final fraction fraction Late fraction product(250 mg/ml) (375 mg/ml) (500 mg/ml) pool (ppm) Residual HCP <7.6 <7.6<7.6 <7.6 ppm (ng/mg product) HCP Log >3.5 >3.5 >3.5 >3.5 Removal Value(LRV) Residual 0.3 Not 0.1 0.6 Protein A determined ppm (ng/mg product)ProA Log 2.9 Not 2.3 2.5 Removal Value (LRV) determined HMW Not Not Not2 fold determined determined determined removal * The impurities in theload were 25,398 ppm of HCP, 99.5 ppm of Protein A, and 2.3% HMW

Example 7.4 Column Runs Under Weak Partitioning Conditions (RobustnessStudies)

To further confirm the performance of the TMAE column in the region ofweak partitioning, several runs were designed varying the pH and NaClconcentration in the load to test process robustness. All runs wereperformed at a load challenge of 250 mg/ml resin. Several MabselectProtein A runs were performed essentially as described in Example 7.2 togenerate the load material for these runs. The only factor varied inthose runs was the sodium chloride concentration in the Protein Aelution, which was varied to match the NaCl concentration in the TMAEload for a particular experiment. The columns were equilibrated withEquil 2 buffers and washed with Wash buffers which had approximately thesame pH and sodium chloride content of the load.

These load conditions are in the weak partitioning region. Batch bindingstudies were used to measure the partition coefficient (Kp). The runsare ranked by the partition coefficients listed in Table 15. The boundproduct was determined by measuring the protein in the column stripusing UV absorbance, and ranges from 7.8-25.3 mg/mL. Protein A, HCP andHMW results from these experiments are also presented in Table 15. Theremoval of all impurities was found to be robust in operating rangeswhich cover 13.5-38.8 mM total chloride and pH 7.8-8.4.

TABLE 15 Process Robustness Studies on Removal of HCP, Protein A, andHMW in WP Mode HCP Protein NaCl Bound in A in Concentration Product Loadload HCP Protein A HMW Recovery (mM) Kp (mg/mL) pH (ppm) (ppm) (LRV)(LRV) (Fold) (%) 38.8 0.26 9.4 7.8 26,391 493.5 3.7 1.8 2.0 92 13.5 0.417.9 7.8 12,821 69.2 3.3 >1.9 1.8 87 27.4 0.50 8 8.0 23,465 252 3.6 2.23.2 91 18.5 0.73 7.8 8.0 21,626 308 3.7 >3.2 2.9 90 23.5 0.80 9.5 8.118,004 343 3.2 >3.2 3.5 94 27.7 0.86 9.5 8.2 24,821 280 3.6 >3.2 2.6 9918.5 1.48 10 8.2 17,669 252 3.7 >3.1 3.9 95 22.0 5.35 25.3 8.4 29,293533 3.6 >2.9 2.3 90 * Impurity levels were 38.5 ppm ProA and 51,943 ppmHCP (Run 1), 8.8 ppm ProA and 25,398 ppm HCP (Run 2). +includes the Cl—ion contribution from NaCl, buffers and titrants

Example 7.5 Summary

From these studies, it can be seen that Protein A removal (LRV) variesstrongly with Kp, while HCP LRV is excellent at all the values of Kp ator above 0.26, but much reduced at Kp=0.17 (under flow-throughconditions). Host cell protein removal is over one log lower forflow-through conditions compared to weak partitioning conditions, evenfor a reduced load challenge. The bound product ranges from 7.8-25 mg/mlfor these weak partitioning conditions on this combination of resin andmonoclonal antibody. The partition coefficient appears to be optimalbetween 0.41<Kp<5.4. It does not appear to be optimal at Kp=0.17 and abound product of 1.4-3.3 mg/mL, the conditions of Example 7.2.

These studies suggest an alternative mode of purifying a polypeptideproduced using rational design media cell culture, which willsignificantly reduce the presence of impurities, high molecular weightaggregates, DNA, host cell proteins, etc.

1. A method of producing a polypeptide in a cell culture comprising: (1)providing a cell culture, comprising: a. cells, comprising a nucleicacid encoding a polypeptide of interest; and b. a desired cell culturemedium, comprising a concentration of an amino acid that is calculatedfor use in cell mass, a concentration of the amino acid that iscalculated for use in cell maintenance, and a concentration of the aminoacid that is calculated for incorporation into the polypeptide ofinterest; and (2) maintaining the cell culture under conditions thatallow expression of the polypeptide of interest.
 2. The method of claim1, wherein both the concentration of an amino acid that is calculatedfor use in cell mass and the concentration of the amino acid that iscalculated for incorporation into the polypeptide of interest aremultiplied by a baseline factor.
 3. The method of claim 1, wherein thedesired cell culture medium, comprises a baseline adjusted amino acidconcentration, A, according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F,wherein X is a concentration of the amino acid that is used per unit ofcell mass, P is a concentration of the amino acid that is used forincorporation into the polypeptide of interest per unit of polypeptidetiter, M is a multiplier for a desired peak cell density of the cellculture, N is a multiplier for a desired concentration of thepolypeptide of interest, Y is a cell maintenance factor, and F is abaseline factor. 4-5. (canceled)
 6. The method of claim 1, wherein themethod comprises providing a starting cell culture medium, wherein thevolume of the starting cell culture medium is about 60-99% of the volumeof a desired cell culture medium volume; and providing a feeding cellculture medium to the cell culture, wherein the volume of the feedingcell culture medium is about 1-40% of the desired cell culture mediumvolume, and wherein the resulting desired cell culture medium comprisesthe concentration of an amino acid that is calculated for use in cellmass, the concentration of the amino acid that is calculated for use incell maintenance, and the concentration of the amino acid that iscalculated for incorporation into the polypeptide of interest.
 7. Themethod of claim 6, wherein the resulting desired cell culture mediumcomprises a baseline-adjusted amino acid concentration, A, according tothe formula A=[(M*X)+(N*P)+(Y*M*X)]*F, wherein X is a concentration ofthe amino acid that is used per unit of cell mass, P is a concentrationof the amino acid that is used for incorporation into the polypeptide ofinterest per unit of polypeptide titer, M is a multiplier for a desiredpeak cell density of the cell culture, N is a multiplier for a desiredconcentration of the polypeptide of interest, Y is a cell maintenancefactor, and F is a baseline factor.
 8. The method of claim 7, whereinthe starting cell culture medium comprises a concentration, B, of theamino acid according to the formula B=[A−(Z*V)]/(1−V), wherein Z is aconcentration of the amino acid in the feeding cell culture medium, andV is a volume of the feeding culture medium as a proportion of thedesired cell culture medium volume.
 9. The method of claim 3, wherein Yis 0 to about 1.5.
 10. The method of claim 3, wherein F is about 1 toabout 1.5. 11-18. (canceled)
 19. The method of claim 3, wherein thecombined concentration of leucine, lysine, threonine, and valine in thedesired cell culture medium is between about 60% and about 80% of theconcentration of the total essential amino acids in the desired cellculture medium.
 20. The method of claim 3, wherein the combinedconcentration of the essential amino acids in the desired cell culturemedium is between about 30% and about 50% of the concentration of thetotal amino acids in the desired cell culture medium.
 21. The method ofclaim 3, wherein the concentration of amino acids in the desired cellculture medium is between about 120 mM and about 350 mM.
 22. The methodof claim 3, wherein the concentration of proline in the cell culture ismaintained at greater than about 1 mM.
 23. The method of claim 3,wherein the concentration of proline in the cell culture is maintainedat greater than about 2 mM. 24-29. (canceled)
 30. A method of cellculture comprising: (1) providing a cell culture, comprising: a. cells;and b. a desired cell culture medium, comprising a concentration of anamino acid that is calculated for use in cell mass and a concentrationof the amino acid that is calculated for use in cell maintenance; and(2) maintaining the cell culture under conditions that allow growth andreplication of the cells in the cell culture.
 31. The method of claim30, wherein the desired cell culture medium, comprises abaseline-adjusted amino acid concentration, A′, according to the formulaA′=[(M*X)+(Y*M*X)]*F, wherein X is a concentration of the amino acidthat is used per unit of cell mass, M is a multiplier for a desired peakcell density of the cell culture, Y is a cell maintenance factor, and Fis a baseline factor. 32-33. (canceled)
 34. The cell culture medium ofclaim 36, comprising a total concentration of amino acids from betweenabout 120 mM and about 350 mM.
 35. (canceled)
 36. A cell culture mediumfor use in the production of a polypeptide of interest, comprising aconcentration of an amino acid that is calculated for use in cell mass,a concentration of the amino acid that is calculated for use in cellmaintenance, and a concentration of the amino acid that is calculatedfor incorporation into the polypeptide of interest.
 37. The cell culturemedium of claim 36, comprising a baseline-adjusted amino acidconcentration, AT, according to the formula A′=[(M*X)+(Y*M*X)]*F,wherein X is a concentration of the amino acid that is used per unit ofcell mass, M is a multiplier for desired peak cell density of the cellculture, Y is a cell maintenance factor, and F is a baseline factor. 38.The cell culture medium of claim 36 for use in the production of apolypeptide of interest, comprising a baseline-adjusted amino acidconcentration, A, according to the formula A=[(M*X)+(N*P)+(Y*M*X)]*F,wherein X is a concentration of the amino acid that is used per unit ofcell mass, P is a concentration of the amino acid that is used forincorporation into the polypeptide of interest per unit of polypeptidetiter, M is a multiplier for desired peak cell density of the cellculture, N is a multiplier for desired concentration of the polypeptideof interest, Y is a cell maintenance factor, and F is a baseline factor.39-41. (canceled)
 42. A method for determining an optimizedconcentration of an amino acid used in a cell culture medium for theproduction of a polypeptide of interest in a cell culture, comprising:(1) determining the amino acid concentration required for the cell massof the cells at a target cell density; (2) determining the amino acidconcentration required to produce the polypeptide of interest at atarget polypeptide titer; (3) determining the amino acid concentrationrequired for cell maintenance of the cells; and (4) adding theconcentrations obtained from step (1), step (2), and step (3) to providean optimized concentration of the amino acid used in the cell culturemedium for the production of the polypeptide of interest. 43-45.(canceled)